Amidated peptides and their deamidated counterparts displayed by HLA-A*02 for use in immunotherapy against different types of cancers

ABSTRACT

The invention relates to a peptide comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 1 to SEQ ID NO: 102, and (ii) a variant sequence thereof which maintains capacity to bind to MHC molecule(s) and/or induce T cells cross-reacting with said variant peptide, or a pharmaceutically acceptable salt thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Application No. 63/084,963,filed 29 Sep. 2020.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.txt)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “Sequence_Listing_2912919-104001_ST25.txt” createdon 29 Sep. 2021 and 42,734 bytes in size) is submitted concurrently withthe instant application, and the entire contents of the Sequence Listingare incorporated herein by reference.

The present invention relates to peptides, proteins, nucleic acids andcells for use in immunotherapeutic methods. In particular, the presentinvention relates to the immunotherapy of cancer. The present inventionfurthermore relates to tumor-associated T cell peptide epitopes, aloneor in combination with other tumor-associated peptides that can forexample serve as active pharmaceutical ingredients of vaccinecompositions that stimulate anti-tumor immune responses, or to stimulateT cells ex vivo and transfer into patients. Peptides bound to moleculesof the major histocompatibility complex (MHC), or peptides as such, canalso be targets of antibodies, soluble T cell receptors, and otherbinding molecules.

The present invention relates to several novel peptide sequences andtheir variants derived from HLA class I molecules of human tumor cellsthat can be used in vaccine compositions for eliciting anti-tumor immuneresponses, or as targets for the development ofpharmaceutically/immunologically active compounds and cells.

BACKGROUND OF THE INVENTION

According to the World Health Organization (WHO), cancer ranged amongthe four major non-communicable deadly diseases worldwide in 2012. Forthe same year, colorectal cancer, breast cancer and respiratory tractcancers were listed within the top 10 causes of death in high incomecountries.

Considering the severe side-effects and expense associated with treatingcancer, there is a need to identify factors that can be used in thetreatment of cancer.

There is also a need to identify factors representing biomarkers forcancer, leading to better diagnosis of cancer, assessment of prognosis,and prediction of treatment success.

Cancer Immunotherapy

Immunotherapy of cancer represents an option of specific targeting ofcancer cells while minimizing side effects. Cancer immunotherapy makesuse of the existence of tumor associated antigens.

The current classification of tumor associated antigens (TAAs) comprisesthe following major groups:

a) Cancer-testis antigens: The first TAAs ever identified that can berecognized by T cells belong to this class, which was originally calledcancer-testis (CT) antigens. Since the cells of testis do not expressclass I and II HLA molecules, these antigens cannot be recognized by Tcells in normal tissues and can therefore be considered asimmunologically tumor specific. Well-known examples for CT antigens arethe MAGE family members and NY-ESO-1.

b) Differentiation antigens: These TAAs are shared between tumors andthe normal tissue from which the tumor arose. Most of the knowndifferentiation antigens are found in melanomas and normal melanocytes.Examples include, but are not limited to, tyrosinase and Melan-A/MART-1for melanoma or PSA for prostate cancer.

c) Overexpressed TAAs: Genes encoding widely expressed TAAs have beendetected in histologically different types of tumors as well as in manynormal tissues, generally with lower expression levels. It is possiblethat many of the epitopes processed and potentially presented by normaltissues are below the threshold level for T cell recognition, whiletheir overexpression in tumor cells can trigger an anticancer responseby breaking previously established tolerance. Prominent examples forthis class of TAAs are Her-2/neu, survivin, telomerase, or WT1.

d) Tumor specific antigens: These unique TAAs arise from mutations ofnormal genes (such as β-catenin, CDK4, etc.). Some of these molecularchanges are associated with neoplastic transformation and/orprogression. Tumor specific antigens are generally able to induce strongimmune responses without bearing the risk for autoimmune reactionsagainst normal tissues. On the other hand, these TAAs are in most casesonly relevant to the exact tumor on which they were identified and areusually not shared between many individual tumors. Tumor specificity (or-association) of a peptide may also arise if the peptide originates froma tumor specific (-associated) exon in case of proteins with tumorspecific (-associated) isoforms.

e) Oncoviral proteins: These TAAs are viral proteins that may play acritical role in the oncogenic process and, because they are foreign(not of human origin), they can evoke a T cell response. Examples ofsuch proteins are the human papilloma type 16 virus proteins, E6 and E7,which are expressed in cervical carcinoma.

Human endogenous retroviruses (HERVs) make up a significant portion(˜8%) of the human genome. These viral elements integrated into thegenome millions of years ago and were since then vertically transmittedthrough generations. The huge majority of HERVs have lost functionalactivity through mutation or truncation, yet some endogenous retrovirus,such as the members of the HERV-K clade, still encode functional genesand have been shown to form retrovirus-like particles. Transcription ofHERV proviruses is epigenetically controlled and remains silenced undernormal physiological conditions. Reactivation and overexpressionresulting in active translation of viral proteins has however beendescribed in certain diseases and especially for different types ofcancer. This tumor-specific expression of HERV derived proteins can beharnessed for different types of cancer immunotherapy.

f) TAAs arising from abnormal post-translational modifications: SuchTAAs may arise from proteins which are neither specific noroverexpressed in tumors but nevertheless become tumor associated bypost-translational processes primarily active in tumors. Examples forthis class arise from altered glycosylation patterns leading to novelepitopes in tumors as for MUC1 or events like protein splicing duringdegradation which may or may not be tumor specific.

T cell based immunotherapy targets peptide epitopes derived fromtumor-associated or tumor specific proteins, which are presented by MHCmolecules. The antigens that are recognized by the tumor specific Tlymphocytes, that is, the epitopes thereof, can be molecules derivedfrom all protein classes, such as enzymes, receptors, transcriptionfactors, etc. which are expressed and as compared to unaltered cells ofthe same origin, usually up-regulated in cells of the respective tumor.

There are two classes of MHC molecules, MHC class I and MHC class II.MHC class I molecules are composed of an alpha heavy chain andbeta-2-microglobulin, MHC class II molecules of an alpha and a betachain. Their three-dimensional conformation results in a binding groove,which is used for non-covalent interaction with peptides.

MHC class I molecules can be found on most nucleated cells. They presentpeptides that result from proteolytic cleavage of predominantlyendogenous proteins, defective ribosomal products (DRIPs) and largerpeptides. However, peptides derived from endosomal compartments orexogenous sources are also frequently found on MHC class I molecules.This non-classical way of class I presentation is referred to ascross-presentation in the literature (Brossart and Bevan, 1997; Rock etal., 1990). MHC class II molecules can be found predominantly onprofessional antigen presenting cells (APCs), and primarily presentpeptides of exogenous or transmembrane proteins that are taken up byAPCs e.g. during endocytosis and are subsequently processed.

Complexes of peptide and MHC class I are recognized by CD8-positive Tcells bearing the appropriate T cell receptor (TCR), whereas complexesof peptide and MHC class II molecules are recognized by CD4-positivehelper T cells bearing the appropriate TCR. It is well known that theTCR, the peptide and the MHC are thereby present in a stoichiometricamount of 1:1:1.

CD4-positive helper T cells play an important role in inducing andsustaining effective responses by CD8-positive cytotoxic T cells. Theidentification of CD4-positive T cell epitopes derived from tumorassociated antigens (TAA) is of great importance for the development ofpharmaceutical products for triggering anti-tumor immune responses. Atthe tumor site, T helper cells, support a cytotoxic T cell (CTL)friendly cytokine milieu and attract effector cells, e.g. CTLs, naturalkiller (NK) cells, macrophages, and granulocytes.

In the absence of inflammation, expression of MHC class II molecules ismainly restricted to cells of the immune system, especially professionalantigen-presenting cells (APC), e.g., monocytes, monocyte-derived cells,macrophages, dendritic cells. In cancer patients, cells of the tumorhave been found to express MHC class II molecules (Dengjel et al.,2006).

Elongated (longer) peptides of the invention can act as MHC class IIactive epitopes.

T helper cells, activated by MHC class II epitopes, play an importantrole in orchestrating the effector function of CTLs in anti-tumorimmunity. T helper cell epitopes that trigger a T helper cell responseof the TH1 type support effector functions of CD8-positive killer Tcells, which include cytotoxic functions directed against tumor cellsdisplaying tumor-associated peptide-MHC complexes on their cellsurfaces. In this way tumor-associated T helper cell peptide epitopes,alone or in combination with other tumor-associated peptides, can serveas active pharmaceutical ingredients of vaccine compositions thatstimulate anti-tumor immune responses.

Since the constitutive expression of HLA class II molecules is usuallylimited to immune cells, the possibility of isolating class II peptidesdirectly from primary tumors was previously not considered possible.However, Dengjel et al. were successful in identifying a number of MHCclass II epitopes directly from tumors (WO 2007/028574, EP 1 760 088 B1,the contents of which are herein incorporated by reference in theirentirety).

For an MHC class I peptide to trigger (elicit) a cellular immuneresponse, it also must bind to an MHC molecule. This process isdependent on the allele of the MHC molecule and specific polymorphismsof the amino acid sequence of the peptide. MHC class I binding peptidesare usually 8-12 amino acid residues in length and usually contain twoconserved residues (“anchors”) in their sequence that interact with thecorresponding binding groove of the MHC molecule. In this way each MHCallele has a “binding motif” determining which peptides can bindspecifically to the binding groove.

In the MHC class I dependent immune reaction, peptides not only have tobe able to bind to certain MHC class I molecules expressed by tumorcells, they subsequently also have to be recognized by T cells bearingspecific TCRs.

For proteins to be recognized by T-lymphocytes as tumor specific orassociated antigens, and to be used in a therapy, particularprerequisites must be fulfilled. The antigen should be expressed mainlyby tumor cells and not, or in comparably small amounts, by normalhealthy tissues. It may be advantageous that a peptide is over-presentedby tumor cells as compared to normal healthy tissues. It is furthermoredesirable that the respective antigen is not only present in a type oftumor, but also in high concentrations (i.e. copy numbers of therespective peptide per cell). Tumor specific and tumor-associatedantigens are often derived from proteins directly involved intransformation of a normal cell to a tumor cell due to their function,e.g. in cell cycle control or suppression of apoptosis. Additionally,downstream targets of the proteins directly causative for atransformation may be up-regulated und thus may be indirectlytumor-associated. Such indirect tumor-associated antigens may also betargets of a vaccination approach (Singh-Jasuja et al., 2004, thecontents of which are herein incorporated by reference in theirentirety). It is essential that epitopes are present in the amino acidsequence of the antigen, in order to ensure that such a peptide(“immunogenic peptide”), being derived from a tumor associated antigen,leads to an in vitro or in vivo T cell response.

TAAs may be a starting point for the development of a T cell basedimmunotherapy. The methods for identifying and characterizing the TAAsare usually based on the use of T cells that can be isolated frompatients or healthy subjects, or they are based on the generation ofdifferential transcription profiles or differential peptide expressionpatterns between tumors and normal tissues. However, the identificationof genes overexpressed in tumor tissues or human tumor cell lines, orselectively expressed in such tissues or cell lines, does not provideprecise information as to the use of the antigens being transcribed fromthese genes in an immune therapy. This is because only an individualsubpopulation of epitopes of these antigens are suitable for such anapplication since a T cell with a corresponding TCR has to be presentand the immunological tolerance for this particular epitope needs to beabsent or minimal.

In case of targeting peptide-MHC by specific TCRs (e.g. soluble TCRs)and antibodies or other binding molecules (scaffolds) according to theinvention, the immunogenicity of the underlying peptides is secondary.In these cases, the presentation is the determining factor.

Deglycosylation and Subsequent Deamidation Generating Tumor AssociatedPeptides

TAAs may not only arise from proteins which are either specific totumors, or overexpressed. They can also arise from abnormalpost-translational modifications (PTMs) of proteins neither specific noroverexpressed in tumors: Such TAAs may nevertheless become tumorassociated by post-translational processes primarily active in tumors.

While post-translational modifications alter and extend the repertoireof the immunopeptidome, they have diverse functions and consequences.Some PTMs hinder the binding of modified peptides to HLA complexes(Andersen et al., 1999) which may contribute to immune evasivestrategies of tumor cells. Other modifications lead to a higher HLAaffinity or increased immunogenicity which has been associated withautoimmune diseases (Arentz-Hansen et al., 2000; McGinty et al., 2015;Sidney et al., 2018; Raposo et al., 2018) but may be exploited forimmunotherapies in malignancies (Zarling et al., 2006; Purcell et al.,2007; Petersen et al., 2009; Cobbold et al., 2013; Marcilla et al.,2014; Lin et al., 2019; Brentville et al., 2020).

Previous PTM analyses identified deamidation as a fairly prevalentmodification of HLA I presented immunopeptides (Han et al., 2011; Mei etal., 2020). This chemical reaction leads to an amino acid conversionfrom asparagine (N) to aspartic acid (D) (Knorre, Kudryashova, andGodovikova 2009; Mei et al., 2020).

N deamidation is enriched in peptides derived from membrane-associatedproteins and is associated with the sequence motif N[X{circumflex over( )}P][ST], where X is any amino acid except proline and is followed byeither a serine (S) or threonine (T) (Han et al., 2011; Cao et al. 2017;Mei et al., 2020). This motif is an established recognition motif forN-glycosyltransferases (Yan and Lennarz 2005; Petersen, Purcell, andRossjohn 2009), linking N deamidation to nascent glycosylation in theendoplasmic reticulum (ER). Mechanistically, proteins or polypeptidesbecome glycosylated during their translation in the ER. After theirexport to the cytoplasm, they become deglycosylated bypeptide-N-glycanase (PNGase). During this hydrolytic degradationprocess, the N residue is also deamidated to D, leading to the terminalchange in the amino acid sequence. After further degradation of theproteins or polypeptides in the proteasome, the peptides arere-transported into the ER. Here, they bind to HLA complexes whichtranslocate to the cell membrane and present the deamidated peptides onthe cell surface (see FIG. 1 ) (Misaghi et al., 2004; Petersen, Purcell,and Rossjohn 2009; Mei et al., 2020). This mechanism potentially allowsT cells to recognize and clear cells with perturbed glycosylation duringinfections but also resulting from altered tumor cell metabolism.

Previous PTM analyses identified deamidation as a fairly prevalentmodification of HLA I presented immunopeptides (Han et al., 2011; Mei etal., 2020). This chemical reaction leads to an amino acid conversionfrom asparagine (N) to aspartic acid (D) (see FIG. 2 ) (Knorre et al.,2009; Mei et al., 2020).

While the aberrant glycosylation in cancer was initially associated withan immune-inhibitory role (Liu and Rabinovich 2005; RodrIguez et al.,2018; De Bousser et al., 2020), cumulative proof shows that it may alsobe exploited for T cell based therapies. Hereby, either theglycoproteins themselves (Posey et al., 2016; Maher et al., 2016;Rodriguez, Schetters, and van Kooyk 2018; De Bousser et al., 2020) orglycosylation-dependent deamidated peptides serve as neo-antigens andmay be targeted. There are some well-described examples of theimmunogenic role of deamidated peptides in literature with the humanimmunodeficiency virus type 1 envelope glycoprotein (GP) (Behrens etal., 2017; Ferris et al., 1999), hepatitis C GP E1 (Selby et al., 1999)and lymphocytic choriomeningitis virus GP 1 (Hudrisier et al., 1999)peptides of pathogens, as well as tyrosinase peptides in melanoma (Mosseet al., 1998; Schaed et al., 2002; Altrich-VanLith et al., 2006;Ostankovitch et al., 2009).

Hence, deamidated peptides and the non-canonical pathway of antigenpresentation described above serve as interesting targets for T cellbased tumor immunotherapy.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to a peptide comprisingan amino acid sequence selected from the group consisting of SEQ ID NO 1to SEQ ID NO 102, and variant sequences thereof which binds to MHCmolecule(s) and/or induces T cells cross-reacting with said variantpeptide, or a pharmaceutically acceptable salt thereof.

The following tables (Tables 1A, 11B, 2A and 2B) show the peptidesaccording to the present invention, their respective SEQ ID NOs, and theprospective source (underlying) genes for these peptides.

TABLE 1A Peptides according to the invention. Note thatthe two neighboring peptides in each line are thewildtype sequence (right column), comprising apotential N-Glycosylation motif, and a mutatedversion devoid of said N-Glycosylation motif(left column), wherein an N (Asn) residue hasbeen replaced by its deamidated variant, D (Asp). SEQ ID DEAMIDATEDSEQ ID NO SEQUENCE NO WT SEQUENCE 1 ILDSTTIEI 103 ILNSTTIEI 2 RLLEGDFSL104 RLLEGNFSL 3 YMDGTMSQV 105 YMNGTMSQV 4 YVWDRTELL 106 YVWNRTELL 5ALPQFFNDV 107 ALPQFFNNV 6 NLYNDLTNV 108 NLYNNLTNV 7 ALADLTGTV 109ALANLTGTV 8 KLAPDLTEL 110 KLAPNLTEL 9 SLDDSLNEL 111 SLNDSLNEL 10FLGEDISNFL 112 FLGENISNFL 11 GLVDGSSVTV 113 GLVNGSSVTV 12 FVDEHTIDI 114FVDEHTINI 13 VLMDGTLKQV 115 VLMNGTLKQV 14 YIYDGKDMSSL 116 YIYDGKNMSSL 15VLDDTLVIF 117 VLNDTLVIF 16 KLISDMGKDVSA 118 KLISDMGKNVSA 17 NVDSTILNL119 NVNSTILNL 18 ILDEAKDISF 120 ILDEAKNISF 19 RLLDTTDVYLL 121RLLNTTDVYLL 20 SLFAYPLPDV 122 SLFAYPLPNV 21 FLNLFDHTL 123 FLNLFNHTL 22HMIDLTIHL 124 HMINLTIHL 23 FLGPIVDL 125 FLGPIVNL 24 QLLGRDISL 126QLLGRNISL 25 FMNDRSFIL 127 FMNNRSFIL 26 ILYDGSNIQL 128 ILYNGSNIQL 27YLFEDISQL 129 YLFENISQL 28 FLLDGTDMFHM 130 FLLNGTDMFHM 29 KLLDLTVRI 131KLLNLTVRI 30 QLMEKVQDV 132 QLMEKVQNV 31 TLIDATWLV 133 TLINATWLV 32ALADLTGTVVNL 134 ALANLTGTVVNL 33 ILFDLTHRV 135 ILFNLTHRV 34 SLLDGSESAKL136 SLLNGSESAKL 35 SLRDGTEVV 137 SLRNGTEVV 36 SLYDFTGEQMAA 138SLYNFTGEQMAA 37 AIDDTAARL 139 AINDTAARL 38 AVDGTDARL 140 AVNGTDARL 39AVFDQTVTVI 141 AVFNQTVTVI 40 FLDQTDETL 142 FLNQTDETL 41 FLDTSTADV 143FLNTSTADV 42 IQDTSHLAV 144 IQNTSHLAV 43 KLLNDLTSI 145 KLLNNLTSI 44LLDATHQI 146 LLNATHQI 45 SLFDDSFSL 147 SLFNDSFSL 46 SLSDVSQAV 148SLSNVSQAV 47 VIDSTLVKV 149 VINSTLVKV 48 AIYHDITGISV 150 AIYHNITGISV 49ALDDYTITF 151 ALDNYTITF 50 ALYDSTRELL 152 ALYNSTRELL 51 HLCNHDVSL 153HLCNHNVSL 52 ILFDDSVFTL 154 ILFNDSVFTL 53 ILTDITGHDV 155 ILTDITGHNV 54KLEERVYDV 156 KLEERVYNV 55 KTWDQSIAL 157 KTWNQSIAL 56 LLDSTAHLL 158LLNSTAHLL 57 SLDISLPNL 159 SLNISLPNL 58 SQDASLLKV 160 SQNASLLKV 59SQTDLSPAL 161 SQTNLSPAL 60 TLDITIVNL 162 TLNITIVNL 61 FISDFTMTI 163FISNFTMTI 62 FLKDVTAQI 164 FLKNVTAQI 63 GLDRTQLVNV 165 GLNRTQLVNV 64ILFDPDNSSAL 166 ILFDPNNSSAL 65 ILLDKSTVL 167 ILLNKSTVL 66 LLDDQTVTF 168LLDNQTVTF 67 LLDTTDVYL 169 LLNTTDVYL 68 RLQDTTIGL 170 RLQNTTIGL 69SLLDENDVSSYL 171 SLLDENNVSSYL 70 SMASFLKDV 172 SMASFLKNV 71 YLGAVFDL 173YLGAVFNL 72 ALDSTNSEL 174 ALNSTNSEL 73 ALMDVSQNV 175 ALMNVSQNV 74KILDFTGPLFL 176 KILNFTGPLFL 75 KLHDTSFCL 177 KLHNTSFCL 76 SLLRDFTLV 178SLLRNFTLV 77 YLHGFDLSL 179 YLHGFNLSL 78 AIDQTITEA 180 AINQTITEA 79ALAGLVYDA 181 ALAGLVYNA 80 ALDSSLQLL 182 ALNSSLQLL 81 AVDKTQTSV 183AVNKTQTSV 82 FLNPDGSDCTL 184 FLNPNGSDCTL 83 FLNVDVSEV 185 FLNVNVSEV 84KMLDETVLV 186 KMLNETVLV 85 LLDITNPVL 187 LLNITNPVL 86 NLADNTILV 188NLANNTILV 87 NMYDLTFHV 189 NMYNLTFHV 88 RLLDETVDVTI 190 RLLNETVDVTI 89SLFIDVTRV 191 SLFINVTRV 90 VLDGTEVNL 192 VLNGTEVNL 91 VLKDGTLVI 193VLKNGTLVI 92 YLADFSHAT 194 YLANFSHAT 93 YLDGTFRLL 195 YLNGTFRLL 94YLWWVNDQSL 196 YLWWVNNQSL 95 KLAPEDLADLTAL 197 KLAPEDLANLTAL 96KLDASLPAL 198 KLNASLPAL 97 KLLDGSQRV 199 KLLNGSQRV 98 NLLADVSTV 200NLLANVSTV 99 NLLDHSSMFL 201 NLLNHSSMFL 100 SLLFQDITL 202 SLLFQNITL 101TLDATVVEL 203 TLNATVVEL 102 WLIDKSMEL 204 WLINKSMEL

TABLE 1B Peptides according to the invention. SEQ ID NO SEQUENCE 205ELAGIGILTV 206 YLLPAIVHI

TABLE 2A Non-deamidated peptides according tothe invention and one exemplary source transcript ID (ENST ID taken fromthe ensemble database https://www.ensembl.org/)from which they derive, however peptides mayfurther originate from other additional oralternative transcripts not listed herein. It isimportant to understand that while the sourcesare indicated for the wildtype variants, theylikewise apply for the deamidated counterpartsas shown in the synopsis of table 1A. SEQ ID NO SEQUENCE TRANSCRIPT ID103 ILNSTTIEI ENST00000423485p915 104 RLLEGNFSL ENST00000440542p117 105YMNGTMSQV ENST00000263321p369 106 YVWNRTELL ENST00000295453p265 107ALPQFFNNV ENST00000304895p413 108 NLYNNLTNV ENST00000251582p1094 109ALANLTGTV ENST00000294785p570 110 KLAPNLTEL ENST00000369682p55 111SLNDSLNEL ENST00000380320p656 112 FLGENISNFL ENST00000319136p273 113GLVNGSSVTV ENST00000294728p414 114 FVDEHTINI ENST00000243077p267 115VLMNGTLKQV ENST00000230036p656 116 YIYDGKNMSSL ENST00000261023p284 117VLNDTLVIF ENST00000326317p262 118 KLISDMGKNVSA ENST00000223026p169 119NVNSTILNL ENST00000220244p418 120 ILDEAKNISF ENST00000421865p1910 121RLLNTTDVYLL ENST00000225719p169 122 SLFAYPLPNV ENST00000306243p360 123FLNLFNHTL ENST00000193322p189 124 HMINLTIHL ENST00000265379p832 125FLGPIVNL ENST00000160262p314 126 QLLGRNISL ENST00000392593p166 127FMNNRSFIL ENST00000477922p551 128 ILYNGSNIQL ENST00000281772p112 129YLFENISQL ENST00000359650p35 130 FLLNGTDMFHM ENST00000261800p2004 131KLLNLTVRI ENST00000219022p133 132 QLMEKVQNV ENST00000264833p67 133TLINATWLV ENST00000305363p220 134 ALANLTGTVVNL ENST00000294785p570 135ILFNLTHRV ENST00000274276p323 136 SLLNGSESAKL ENST00000225276p127 137SLRNGTEVV ENST00000258526p146 138 SLYNFTGEQMAA ENST00000261023p293 139AINDTAARL ENST00000251081p627 140 AVNGTDARL ENST00000278937p37 141AVFNQTVTVI ENST00000244763p188 142 FLNQTDETL ENST00000227918p66 143FLNTSTADV ENST00000254508p924 144 IQNTSHLAV ENST00000243776p359 145KLLNNLTSI ENST00000258341p646 146 LLNATHQI ENST00000294785p53 147SLFNDSFSL ENST00000555868p168 148 SLSNVSQAV ENST00000370454p67 149VINSTLVKV ENST00000256509p836 150 AIYHNITGISV ENST00000368507p329 151ALDNYTITF ENST00000254508p1036 152 ALYNSTRELL ENST00000238682p71 153HLCNHNVSL ENST00000388922p93 154 ILFNDSVFTL ENST00000287497p1072 155ILTDITGHNV ENST00000265662p1670 156 KLEERVYNV ENST00000262101p214 157KTWNQSIAL ENST00000367796p2211 158 LLNSTAHLL ENST00000224721p2098 159SLNISLPNL ENST00000326654p27 160 SQNASLLKV ENST00000240618p129 161SQTNLSPAL ENST00000367796p1268 162 TLNITIVNL ENST00000397459p40 163FISNFTMTI ENST00000218436p80 164 FLKNVTAQI ENST00000361866p801 165GLNRTQLVNV ENST00000264036p416 166 ILFDPNNSSAL ENST00000305231p62 167ILLNKSTVL ENST00000271588p3724 168 LLDNQTVTF ENST00000346145p529 169LLNTTDVYL ENST00000225719p170 170 RLQNTTIGL ENST00000538324p109 171SLLDENNVSSYL ENST00000314191p3305 172 SMASFLKNV ENST00000326303p240 173YLGAVFNL ENST00000367721p244 174 ALNSTNSEL ENST00000355540p1393 175ALMNVSQNV ENST00000303354p1372 176 KILNFTGPLFL ENST00000240487p298 177KLHNTSFCL ENST00000478191p59 178 SLLRNFTLV ENST00000262607p181 179YLHGFNLSL ENST00000261800p325 180 AINQTITEA ENST00000258341p1393 181ALAGLVYNA ENST00000371579p308 182 ALNSSLQLL ENST00000380320p792 183AVNKTQTSV ENST00000281171p731 184 FLNPNGSDCTL ENST00000358387p202 185FLNVNVSEV ENST00000237019p79 186 KMLNETVLV ENST00000271588p1926 187LLNITNPVL ENST00000313984p155 188 NLANNTILV ENST00000305139p343 189NMYNLTFHV ENST00000339381p429 190 RLLNETVDVTI ENST00000266581p370 191SLFINVTRV ENST00000289547p134 192 VLNGTEVNL ENST00000258796p349 193VLKNGTLVI ENST00000217939p1907 194 YLANFSHAT ENST00000355396p420 195YLNGTFRLL ENST00000303375p1132 196 YLWWVNNQSL ENST00000006724p177 197KLAPEDLANLTAL ENST00000360658p234 198 KLNASLPAL ENST00000369762p168 199KLLNGSQRV ENST00000236040p464 200 NLLANVSTV ENST00000268793p355 201NLLNHSSMFL ENST00000612687p765 202 SLLFQNITL ENST00000221954p106 203TLNATVVEL ENST00000317620p268 204 WLINKSMEL ENST00000374736p518

TABLE 2B Peptides according to the invention and oneexemplary source transcript ID (ENST ID taken fromthe ensemble database https://www.ensembl.org/)from which they derive, however peptides mayfurther originate from other additional oralternative transcripts not listed herein. SEQ ID NO SEQUENCETRANSCRIPT ID 205 ELAGIGILTV ENST00000381471p26 206 YLLPAIVHIENST00000225792p148

The present invention furthermore generally relates to the peptidesaccording to the present invention for use in the treatment ofproliferative diseases. Proliferative diseases in the context are, forexample, acute myeloid leukemia, breast cancer, cholangiocellularcarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladdercancer, glioblastoma, gastric cancer, gastro-esophageal junction cancer,hepatocellular carcinoma, head and neck squamous cell carcinoma,melanoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovariancancer, esophageal cancer, pancreatic cancer, prostate cancer, renalcell carcinoma, small cell lung cancer, urinary bladder carcinoma, anduterine endometrial cancer.

Particularly preferred are the peptides—alone or incombination—according to the present invention selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 102.

Thus, another aspect of the present invention relates to the use of thepeptides according to the present invention for the—preferablycombined—treatment of a proliferative disease.

The present invention furthermore relates to peptides according to thepresent invention that have the ability to bind to an MHC molecule classI or—in an elongated form, such as a length-variant—MHC class II.

The present invention further relates to elongated peptides, which afteradministration (e.g. as vaccine) can be processed intracellularly,leading to shorter peptides consisting or consisting essentially of theamino acid sequences according to SEQ ID NO: 1 to SEQ ID NO: 102 whichis then presented by HLA on the cell surface.

The present invention further relates to the peptides according to thepresent invention wherein said peptides (each) consist or consistessentially of an amino acid sequence according to SEQ ID NO: 1 to SEQID NO: 102.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide includes non-peptide bonds.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is part of a fusion protein, inparticular fused to the N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii) or fused to (or into thesequence of) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the present invention. The present inventionfurther relates to the nucleic acid according to the present inventionthat is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing and/or expressing a nucleic acid according to the presentinvention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use in thetreatment of diseases and in medicine, in particular in the treatment ofcancer.

The present invention further relates to antibodies that are specificagainst the peptides according to the present invention or complexes ofsaid peptides according to the present invention with MHC, and methodsof making these.

The present disclosure may also relate to methods of producing anantibody specifically binding to an MHC class I molecule complexed withan peptide comprising, consisting of, or consisting essentially of anamino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 102,including immunizing genetically engineered non-human mammal containingcells expressing said MHC class I molecule with a soluble form of an MHCclass I molecule complexed with a peptide consisting or consistingessentially of an amino acid sequence according to SEQ ID NO: 1 to SEQID NO: 102; isolating mRNA molecules from antibody producing cells ofsaid non-human mammal; producing a phage display library displayingprotein molecules encoded by said mRNA molecules; and isolating at leastone phage from said phage display library, wherein said at least onephage displays said antibody specifically binding to said MHC class Imolecule complexed with a peptide comprising, consisting of, orconsisting essentially of an amino acid sequence according to SEQ ID NO:1 to SEQ ID NO: 102. In another aspect, the antibody may be a monoclonalantibody.

In an aspect, the antibody may bind to said MHC class I moleculecomplexed with an antigen comprising, consisting of, or consistingessentially of an amino acid sequence according to SEQ ID NO: 1 to SEQID NO: 102 with a binding affinity (K_(d)) of <100 nM, more preferably<50 nM, more preferably <10 nM, more preferably <1 nM, more preferably<0.1 nM, more preferably <0.01 nM.

In another aspect, methods of producing an antibody may further includehumanizing the antibody. In aspects, methods of producing an antibodymay further include conjugating the antibody with a toxin. In anotheraspect, methods of producing an antibody may further include conjugatingthe antibody with an immune stimulating domain.

In an aspect, methods of producing an antibody may further includemodifying the antibody in the form of a bispecific antibody. In anotheraspect, methods of producing an antibody may further include modifyingthe antibody in the form of a chimeric antibody. In another aspect,methods of producing an antibody may further include modifying theantibody in the form of an Fv. In an aspect, methods of producing anantibody may further include modifying the antibody in the form of aFab. In another aspect, methods of producing an antibody may furtherinclude modifying the antibody in the form of a Fab′. In another aspect,methods of producing an antibody may further include labeling theantibody with a radionucleotide, which may be selected from the groupconsisting of ¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P, and ³⁵S. In anotheraspect, the non-human mammal may be a mouse.

The present invention further relates to TCRs, in particular solubleTCRs and cloned TCRs engineered into autologous or allogeneic T cells,and methods of making these, as well as NK cells or other cells bearingsaid TCR or cross-reacting with said TCRs. The soluble TCR may have abinding affinity (K_(d))<100 nM, more preferably <50 nM, more preferably<10 nM, more preferably <1 nM, more preferably <0.1 nM, more preferably<0.01 nM. Whereas the cloned cell based TCR may have a binding affinity(K_(d))<50 μM, more preferably <25 μM, more preferably <10 μM, morepreferably <1 μM, more preferably <0.1 μM.

The antibodies and TCRs are additional embodiments of theimmunotherapeutic use of the peptides according to the invention athand.

The present invention further relates to a host cell comprising anucleic acid according to the present invention or an expression vectoras described before. The present invention further relates to the hostcell according to the present invention that is an antigen presentingcell, and preferably is a dendritic cell.

The present invention further relates to said method according to thepresent invention, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell.

The present invention further relates to the method according to thepresent invention, wherein the antigen-presenting cell comprises anexpression vector capable of expressing said peptide containing SEQ IDNO: 1 to SEQ ID NO: 102.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellselectively recognizes a cell which expresses a polypeptide comprisingan amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient whose target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprises administering an effective number of T cells to thepatient as produced according to the present invention.

The present invention further relates to the use of any peptide asdescribed, the nucleic acid according to the present invention, theexpression vector according to the present invention, the cell accordingto the present invention, the activated T lymphocyte, the TCR or theantibody or other peptide and/or peptide-MHC binding molecules accordingto the present invention as a medicament or in the manufacture of amedicament. Preferably, said medicament is active against cancer.Preferably, said medicament is a cellular therapy, a vaccine or aprotein based on a soluble TCR or antibody.

The present invention further relates to a use according to the presentinvention, wherein said cancers are acute myeloid leukemia, breastcancer, cholangiocellular carcinoma, chronic lymphocytic leukemia,colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer,gastro-esophageal junction cancer, hepatocellular carcinoma, head andneck squamous cell carcinoma, melanoma, non-Hodgkin lymphoma, non-smallcell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, small cell lung cancer, urinarybladder carcinoma, and uterine endometrial cancer, and preferably acutemyeloid leukemia, breast cancer, cholangiocellular carcinoma, chroniclymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, gastro-esophageal junction cancer,hepatocellular carcinoma, head and neck squamous cell carcinoma,melanoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovariancancer, esophageal cancer, pancreatic cancer, prostate cancer, renalcell carcinoma, small cell lung cancer, urinary bladder carcinoma, anduterine endometrial cancer cells.

The present invention further relates to biomarkers based on thepeptides according to the present invention, herein called “targets”that can be used in the diagnosis of cancer, preferably acute myeloidleukemia, breast cancer, cholangiocellular carcinoma, chroniclymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, gastro-esophageal junction cancer,hepatocellular carcinoma, head and neck squamous cell carcinoma,melanoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovariancancer, esophageal cancer, pancreatic cancer, prostate cancer, renalcell carcinoma, small cell lung cancer, urinary bladder carcinoma, anduterine endometrial cancer. The marker can be over-presentation of thepeptide(s) themselves, or overexpression of the corresponding gene(s).The markers may also be used to predict the probability of success of atreatment, preferably an immunotherapy, and most preferred animmunotherapy targeting the same target that is identified by thebiomarker. For example, an antibody or soluble TCR can be used to stainsections of the tumor to detect the presence of a peptide of interest incomplex with MHC.

Optionally, the antibody carries a further effector function such as animmune stimulating domain or toxin.

The present invention also relates to the use of these novel targets inthe context of cancer treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-7E depict embodiments as described herein.

DETAILED DESCRIPTION OF THE INVENTION

Stimulation of an immune response is dependent upon the presence ofantigens recognized as foreign by the host immune system. The discoveryof the existence of tumor associated antigens has raised the possibilityof using a host's immune system to intervene in tumor growth. Variousmechanisms of harnessing both the humoral and cellular arms of theimmune system are currently being explored for cancer immunotherapy.

Specific elements of the cellular immune response are capable ofspecifically recognizing and destroying tumor cells. The isolation of Tcells from tumor-infiltrating cell populations or from peripheral bloodsuggests that such cells play an important role in natural immunedefense against cancer. CD8-positive T cells in particular, whichrecognize MHC class I molecules bearing peptides of usually 8 to 12amino acid residues derived from proteins or defect ribosomal products(DRIPS) located in the cytosol, play an important role in this response.The MHC molecules of the human are also designated as humanleukocyte-antigens (HLA).

As used herein and except as noted otherwise all terms are defined asgiven below.

The term “T cell response” means the specific proliferation andactivation of effector functions induced by a peptide in vitro or invivo. For MHC class I restricted cytotoxic T cells, effector functionsmay be lysis of peptide pulsed, peptide-precursor pulsed or naturallypeptide-presenting target cells, secretion of cytokines, preferablyInterferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion ofeffector molecules, preferably granzymes or perforins induced bypeptide, or degranulation.

The term “peptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carboxyl groups of the adjacent amino acids.

Furthermore, the term “peptide” shall include salts of a series of aminoacid residues, connected one to the other typically by peptide bondsbetween the alpha-amino and carboxyl groups of the adjacent amino acids.Preferably, the salts are pharmaceutically acceptable salts of thepeptides, such as, for example, the chloride or acetate(trifluoroacetate) salts. It has to be noted that the salts of thepeptides according to the present invention differ substantially fromthe peptides in their state(s) in vivo, as the peptides are not in theform of salts or associated with counterions in vivo.

The term “peptide” shall also include “oligopeptide”. The term“oligopeptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carboxyl groups of the adjacent amino acids. Thelength of the oligopeptide is not critical to the invention, as long asthe correct epitope or epitopes are maintained therein.

The term “polypeptide” designates a series of amino acid residues,connected one to the other typically by peptide bonds between thealpha-amino and carboxyl groups of the adjacent amino acids. The lengthof the polypeptide is not critical to the invention as long as thecorrect epitopes are maintained. In contrast to the terms peptide oroligopeptide, the term polypeptide is meant to refer to moleculescontaining more than about 30 amino acid residues.

A peptide, oligopeptide, protein or polynucleotide coding for such amolecule is “immunogenic” (and thus is an “immunogen” within the presentinvention), if it is capable of inducing an immune response. In the caseof the present invention, immunogenicity is more specifically defined asthe ability to induce a T cell response. Thus, an “immunogen” would be amolecule that is capable of inducing an immune response, and in the caseof the present invention, a molecule capable of inducing a T cellresponse. In another aspect, the immunogen can be the peptide, thecomplex of the peptide with MHC, oligopeptide, and/or protein that isused to raise specific antibodies or TCRs against it.

A class I T cell “epitope” requires a short peptide that is bound to aclass I MHC molecule, forming a ternary complex (MHC class I alphachain, beta-2-microglobulin, and peptide) that can be recognized by a Tcell bearing a matching T cell receptor binding to the MHC-peptidecomplex with appropriate affinity. Peptides binding to MHC class Imolecules are typically 8-12 amino acids in length, and most typically 9amino acids in length.

In humans there are three different genetic loci that encode MHC class Imolecules (the MHC molecules of the human are also designated humanleukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02,and HLA-B*07 are examples of different MHC class I alleles that can beexpressed from these loci.

The peptides of the invention, preferably when included into a vaccineof the invention as described herein bind to HLA-A*02. A vaccine mayalso include pan-binding MHC class II peptides. Therefore, the vaccineof the invention can be used to treat cancer in patients that areHLA-A*02-positive, whereas no selection for MHC class II allotypes isnecessary due to the pan-binding nature of these peptides.

If HLA-A*02 peptides of the invention are combined with peptides bindingto another allele, for example HLA-A*24, a higher percentage of anypatient population can be treated compared with addressing either MHCclass I allele alone. While in most populations less than 50% ofpatients could be addressed by either allele alone, a vaccine comprisingHLA-A*24 and HLA-A*02 epitopes can treat at least 60% of patients in anyrelevant population. Specifically, the following percentages of patientswill be positive for at least one of these alleles in various regions:USA 61%, Western Europe 62%, China 75%, South Korea 77%, Japan 86%(calculated from allelefrequencies.net).

In a preferred embodiment, the term “nucleotide sequence” refers to aheteropolymer of deoxyribonucleotides.

The nucleotide sequence coding for a particular peptide, oligopeptide,or polypeptide may be naturally occurring or they may be syntheticallyconstructed. Generally, DNA segments encoding the peptides,polypeptides, and proteins of this invention are assembled from cDNAfragments and short oligonucleotide linkers, or from a series ofoligonucleotides, to provide a synthetic gene that is capable of beingexpressed in a recombinant transcriptional unit comprising regulatoryelements derived from a microbial or viral operon.

As used herein the term “a nucleotide coding for (or encoding) apeptide” refers to a nucleotide sequence coding for the peptideincluding artificial (man-made) start and stop codons compatible for thebiological system the sequence is to be expressed by, for example, adendritic cell or another cell system useful for the production of TCRs.

As used herein, reference to a nucleic acid sequence includes bothsingle stranded and double stranded nucleic acid. Thus, for example forDNA, the specific sequence, unless the context indicates otherwise,refers to the single strand DNA of such sequence, the duplex of suchsequence with its complement (double stranded DNA) and the complement ofsuch sequence.

The term “coding region” refers to that portion of a gene which eithernaturally or normally codes for the expression product of that gene inits natural genomic environment, i.e., the region coding in vivo for thenative expression product of the gene.

The coding region can be derived from a non-mutated (“normal”), mutatedor altered gene, or can even be derived from a DNA sequence, or gene,wholly synthesized in the laboratory using methods well known to thoseof skill in the art of DNA synthesis.

The term “expression product” means the polypeptide or protein that isthe natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid(s).

The term “fragment”, when referring to a coding sequence, means aportion of DNA comprising less than the complete coding region, whoseexpression product retains essentially the same biological function oractivity as the expression product of the complete coding region.

The term “DNA segment” refers to a DNA polymer, in the form of aseparate fragment or as a component of a larger DNA construct, which hasbeen derived from DNA isolated at least once in a substantially pureform, i.e., free of contaminating endogenous materials and in a quantityor concentration enabling identification, manipulation, and recovery ofthe segment and its component nucleotide sequences by standardbiochemical methods, for example, by using a cloning vector. Suchsegments are provided in the form of an open reading frame uninterruptedby internal non-translated sequences, or introns, which are typicallypresent in eukaryotic genes. Sequences of non-translated DNA may bepresent downstream from the open reading frame, where the same do notinterfere with manipulation or expression of the coding regions.

The term “a pharmaceutically acceptable salt” refers to a derivative ofthe disclosed peptides wherein the peptide is modified by making acid orbase salts of the agent. For example, acid salts are prepared from thefree base (typically wherein the neutral form of the drug has a neutral—NH₂ group) involving reaction with a suitable acid. Suitable acids forpreparing acid salts include both organic acids, e.g., acetic acid,propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid,malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid,citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethane sulfonic acid, p-toluenesulfonic acid, salicylicacid, and the like, as well as inorganic acids, e.g., hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid phosphoric acid and thelike. Conversely, preparation of basic salts of acid moieties which maybe present on a peptide are prepared using a pharmaceutically acceptablebase such as sodium hydroxide, potassium hydroxide, ammonium hydroxide,calcium hydroxide, trimethylamine or the like. In a preferredembodiment, the pharmaceutical compositions comprise the peptides assalts of acetic acid (acetates), trifluor acetates or hydrochloric acid(chlorides).

The term “promoter” means a region of DNA involved in binding of RNApolymerase to initiate transcription.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment, if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polynucleotides, and recombinant or immunogenic polypeptides,disclosed in accordance with the present invention may also be in“purified” form. The term “purified” does not require absolute purity,rather it is intended as a relative definition, and can includepreparations that are highly purified or preparations that are onlypartially purified, as those terms are understood by those of skill inthe relevant art. For example, individual clones isolated from a cDNAlibrary have been conventionally purified to electrophoretichomogeneity. Purification of starting material or natural material to atleast one order of magnitude, preferably two or three orders, and morepreferably four or five orders of magnitude is expressly contemplated.Furthermore, a claimed polypeptide which has a purity of preferably99.999%, or at least 99.99% or 99.9%; and even desirably 99% by weightor greater is expressly encompassed.

The nucleic acids and polypeptides expression products disclosedaccording to the present invention, as well as expression vectorscontaining such nucleic acids and/or such polypeptides, may be in“enriched form”. As used herein, the term “enriched” means that theconcentration of the material is at least about 2, 5, 10, 100, or 1000times its natural concentration (for example), advantageously 0.01%, byweight, preferably at least about 0.1% by weight. Enriched preparationsof about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. Thesequences, constructs, vectors, clones, and other materials comprisingthe present invention can advantageously be in an enriched or isolatedform. The term “active fragment” means a fragment, usually of a peptide,polypeptide or nucleic acid sequence, that generates an immune response(i.e., has immunogenic activity) when administered, alone or optionallywith a suitable adjuvant or in a vector, to an animal, such as a mammal,for example, mouse, rat, llama, sheep, goat, dog, or horse, and alsoincluding a human, such immune response taking the form of stimulating aT cell response within the recipient animal, such as a human.Alternatively, the “active fragment” may also be used to induce a T cellresponse in vitro.

As used herein, the terms “portion”, “segment” and “fragment”, when usedin relation to polypeptides, refer to a continuous sequence of residues,such as amino acid residues, which sequence forms a subset of a largersequence. For example, if a polypeptide were subjected to treatment withany of the common endopeptidases, such as trypsin or chymotrypsin, theoligopeptides resulting from such treatment would represent portions,segments or fragments of the starting polypeptide. When used in relationto polynucleotides, these terms refer to the products produced bytreatment of said polynucleotides with any endonucleases.

In accordance with the present invention, the term “percent identity” or“percent identical”, when referring to a sequence, means that a sequenceis compared to a claimed or described sequence after alignment of thesequence to be compared (the “compared sequence”) with the described orclaimed sequence (the “reference sequence”). The percent identity isthen determined according to the following formula:percent identity=100[1−(C/R)]

wherein C is the number of differences between the reference sequenceand the compared sequence over the length of alignment between thereference sequence and the compared sequence, wherein

-   -   (i) each base or amino acid in the reference sequence that does        not have a corresponding aligned base or amino acid in the        compared sequence and    -   (ii) each gap in the reference sequence and    -   (iii) each aligned base or amino acid in the reference sequence        that is different from an aligned base or amino acid in the        compared sequence, constitutes a difference and    -   (iiii) the alignment has to start at position 1 of the aligned        sequences, and R is the number of bases or amino acids in the        reference sequence over the length of the alignment with the        compared sequence with any gap created in the reference sequence        also being counted as a base or amino acid.

If an alignment exists between the compared sequence and the referencesequence for which the percent identity as calculated above is aboutequal to or greater than a specified minimum percent identity, then thecompared sequence has the specified minimum percent identity to thereference sequence even though alignments may exist in which the hereinabove calculated percent identity is less than the specified percentidentity.

As mentioned above, the present invention thus provides a peptidecomprising a sequence that is selected from the group of consisting ofSEQ ID NO: 1 to SEQ ID NO: 102 or a variant thereof that will induce Tcells cross-reacting with said peptide. The peptides of the inventionhave the ability to bind to an MHC molecule class I or elongatedversions of said peptides to class II.

In the present invention, the term “homologous” refers to the degree ofidentity (see percent identity above) between sequences of two aminoacid sequences, i.e., peptide or polypeptide sequences. Theaforementioned “homology” is determined by comparing two sequencesaligned under optimal conditions over the sequences to be compared. Sucha sequence homology can be calculated by creating an alignment using,for example, the ClustalW algorithm. Commonly available sequenceanalysis software, more specifically, Vector NTI, GENETYX or other toolsare provided by public databases.

A person skilled in the art will be able to assess, whether T cellsinduced by a variant of a specific peptide will be able to cross-reactwith the peptide itself (see, for example, Appay et al., 2006;Colombetti et al., 2006; Fong et al., 2001; Zaremba et al., 1997, thecontents which are incorporated by reference in their entireties).

By a “variant” of the given amino acid sequence the inventors mean thatthe side chains of, for example, one or two of the amino acid residuesare altered (for example by replacing them with the side chain ofanother naturally occurring amino acid residue or some other side chain)such that the peptide is still able to bind to an HLA molecule insubstantially the same way as a peptide consisting of the given aminoacid sequence in SEQ ID NO: 1 to SEQ ID NO: 102. For example, a peptidemay be modified so that it at least maintains, if not improves, theability to interact with and bind to the binding groove of a suitableMHC molecule, such as HLA-A*02, and in that way, it at least maintains,if not improves, the ability to bind to the TCR of activated T cells.

These T cells can subsequently cross-react with cells and kill cellsthat express a polypeptide that contains the natural amino acid sequenceof the cognate peptide as defined in the aspects of the invention. Ascan be derived from the scientific literature and databases (Rammenseeet al., 1999; Godkin et al., 1997, which are incorporated by referencein their entireties), certain positions of HLA binding peptides aretypically anchor residues forming a core sequence fitting to the bindingmotif of the HLA molecule, which is defined by polar, electrophysical,hydrophobic and spatial properties of the polypeptide chainsconstituting the binding groove. Thus, one skilled in the art would beable to modify the amino acid sequences set forth in SEQ ID NO: 1 to SEQID NO: 102, by maintaining the known anchor residues, and would be ableto determine whether such variants maintain the ability to bind MHCclass I or II molecules. The variants of the present invention retainthe ability to bind to the TCR of activated T cells, which cansubsequently cross-react with and kill cells that express a polypeptidecontaining the natural amino acid sequence of the cognate peptide asdefined in the aspects of the invention.

The original (unmodified) peptides as disclosed herein can be modifiedby the substitution of one or more residues at different, possiblyselective, sites within the peptide chain, if not otherwise stated.Preferably those substitutions are located at the end of the amino acidchain. Such substitutions may be of a conservative nature, for example,where one amino acid is replaced by an amino acid of similar structureand characteristics, such as where a hydrophobic amino acid is replacedby another hydrophobic amino acid. Even more conservative would be thereplacement of amino acids of the same or similar size and chemicalnature, such as where leucine is replaced by isoleucine. In studies ofsequence variations in families of naturally occurring homologousproteins, certain amino acid substitutions are more often tolerated thanothers, and these often show similarities in size, charge, polarity, andhydrophobicity between the original amino acid and its replacement, andsuch is the basis for defining “conservative substitutions”.

Conservative substitutions are herein defined as exchanges within one ofthe following five groups: Group 1—small aliphatic, nonpolar, orslightly polar residues (Ala, Ser, Thr, Pro, Gly); Group 2—polar,negatively charged residues and their amides (Asp, Asn, Glu, Gln); Group3—polar, positively charged residues (His, Arg, Lys); Group 4—large,aliphatic, nonpolar residues (Met, Leu, Ile, Val, Cys); and Group5—large, aromatic residues (Phe, Tyr, Trp).

In an aspect, conservative substitutions may include those, which aredescribed by Dayhoff in “The Atlas of Protein Sequence and Structure.Vol. 5”, Natl. Biomedical Research, the contents of which areincorporated by reference in their entirety. For example, in an aspect,amino acids, which belong to one of the following groups, can beexchanged for one another, thus, constituting a conservative exchange:Group 1: alanine (A), proline (P), glycine (G), asparagine (N), serine(S), threonine (T); Group 2: cysteine (C), serine (S), tyrosine (Y),threonine (T); Group 3: valine (V), isoleucine (I), leucine (L),methionine (M), alanine (A), phenylalanine (F); Group 4: lysine (K),arginine (R), histidine (H); Group 5: phenylalanine (F), tyrosine (Y),tryptophan (W), histidine (H); and Group 6: aspartic acid (D), glutamicacid (E). In an aspect, a conservative amino acid substitution may beselected from the following of T→A, G→A, A→I, T→V, A→M, T→I, A→V, T→G,and/or T→S.

In an aspect, a conservative amino acid substitution may include thesubstitution of an amino acid by another amino acid of the same class,for example, (1) nonpolar: Ala, Val, Leu, Ile, Pro, Met, Phe, Trp; (2)uncharged polar: Gly, Ser, Thr, Cys, Tyr, Asn, Gln; (3) acidic: Asp,Glu; and (4) basic: Lys, Arg, His. Other conservative amino acidsubstitutions may also be made as follows: (1) aromatic: Phe, Tyr, His;(2) proton donor: Asn, Gln, Lys, Arg, His, Trp; and (3) proton acceptor:Glu, Asp, Thr, Ser, Tyr, Asn, Gln (see, for example, U.S. Pat. No.10,106,805, the contents of which are incorporated by reference in theirentirety).

In another aspect, conservative substitutions may be made in accordancewith table 3. Methods for predicting tolerance to protein modificationmay be found in literature (for example, Guo et al., 2004 the contentsof which are incorporated by reference in their entirety).

TABLE 3 List of conservative amino acid substitutions. Original residueConservative substitutions (others are known in the art) Ala Ser, Gly,Cys Arg Lys, Gln, His Asn Gln, His, Glu, Asp Asp Glu, Asn, Gln Cys Ser,Met, Thr Gln Asn, Lys, Glu, Asp, Arg Glu Asp, Asn, Gln Gly Pro, Ala, SerHis Asn, Gln, Lys Ile Leu, Val, Met, Ala Leu Ile, Val, Met, Ala Lys Arg,Gln, His Met Leu, Ile, Val, Ala, Phe Phe Met, Leu, Tyr, Trp, His SerThr, Cys, Ala Thr Ser, Val, Ala Trp Tyr, Phe Tyr Trp, Phe, His Val Ile,Leu, Met, Ala, Thr

In another aspect, substitutions may be those shown in table 4. If suchsubstitutions result in a change in biological activity, then moresubstantia changes, denominated “exemplary substitutions” in table 4,may be introduced and the products screened if needed.

TABLE 4 Exemplary amino acid substitutions. Original residue Exemplarysubstitutions (other are known in the art) Ala Val, Leu, Ile Arg Lys,Gln, Asn Asn Gln, His, Asp, Lys, Arg Asp Glu, Asn Cys Ser, Ala Gln Asn,Glu Glu Asp, Gln Gly Ala His Asn, Gln, Lys, Arg Ile Leu, Val, Met, Ala,Phe, Norleucin Leu Norleucin, Ile, Val, Met, Ala, Phe Lys Arg, Gln, AsnMet Leu, Phe, Ile Phe Leu, Val, Ile, Ala, Tyr Pro Ala Ser Thr Thr Ser,Ala Trp Tyr, Phe Tyr Trp, Phe, Thr, Ser Val Ile, Leu, Met, Phe, Ala,Norleucin

Of course, such substitutions may involve structures other than thecommon L-amino acids. Thus, D-amino acids might be substituted for theL-amino acids commonly found in the antigenic peptides of the inventionand yet still be encompassed by the disclosure herein. In addition,non-standard amino acids (i.e., other than the common naturallyoccurring proteinogenic amino acids) may also be used for substitutionpurposes to produce immunogens and immunogenic polypeptides according tothe present invention.

If substitutions at more than one position are found to result in apeptide with substantially equivalent or greater antigenic activity asdefined below, then combinations of those substitutions will be testedto determine if the combined substitutions result in additive orsynergistic effects on the antigenicity of the peptide.

A peptide consisting essentially of the amino acid sequence as indicatedherein can have one or two non-anchor amino acids (see below regardingthe anchor motif) exchanged without its ability to bind to an MHCmolecule class I or II being substantially changed or negativelyaffected, when compared to the non-modified peptide. In anotherembodiment, in a peptide consisting essentially of the amino acidsequence as indicated herein, one or two amino acids can be exchangedwith their conservative exchange partners (see herein below) without theability to bind to an MHC molecule class I or II being substantiallychanged, or is negatively affected, when compared to the non-modifiedpeptide.

The amino acid residues that do not substantially contribute tointeractions with the T cell receptor can be modified by replacementwith other amino acids whose incorporation does not substantially affectT cell reactivity and does not eliminate binding to the relevant MHC.Thus, apart from the proviso given, the peptide of the invention may beany peptide (by which term the inventors include oligopeptide orpolypeptide), which includes the amino acid sequences, or a portion orvariant thereof as given.

Longer (elongated) peptides may also be suitable. It is possible thatMHC class I epitopes, although usually between 8 and 12 amino acidslong, are generated by peptide processing from longer peptides orproteins that include the actual epitope. It is preferred that theresidues that flank the actual epitope are residues that do notsubstantially affect proteolytic cleavage necessary to expose the actualepitope during processing.

The peptides of the invention can be elongated by up to four aminoacids, that is 1, 2, 3 or 4 amino acids can be added to either end inany combination between 4:0 and 0:4. Combinations of the elongationsaccording to the invention can be found in table 5.

TABLE 5 Combinations of the elongations of peptides of the inventionC-terminus N-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0or 1 or 2 or 3 or 4 N-terminus C-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4

The amino acids for the elongation/extension can be the peptides of theoriginal sequence of the protein or any other amino acid(s). Theelongation can be used to enhance the stability or solubility of thepeptides.

Thus, the epitopes of the present invention may be identical tonaturally occurring tumor-associated or tumor specific epitopes or mayinclude epitopes that differ by no more than four residues from thereference peptide, as long as they have substantially identicalantigenic activity.

In an alternative embodiment, the peptide is elongated on either or bothsides by more than 4 amino acids, preferably to a total length of up to30 amino acids. This may lead to MHC class II binding peptides. Bindingto MHC class II can be tested by methods known in the art.

Accordingly, the present invention provides peptides and variants of MHCclass I epitopes, wherein the peptide or variant has an overall lengthof between 8 and 100, preferably between 8 and 30, and most preferredbetween 8 and 12, namely 8, 9, 10, 11, 12 amino acids, in case of theelongated class II binding peptides the length can also be 13, 14, 15,16, 17, 18, 19, 20, 21 or 22 amino acids.

Of course, the peptide or variant according to the present inventionwill have the ability to bind to an MHC molecule class I or II. Bindingof a peptide or a variant to an MHC complex may be tested by methodsknown in the art.

Preferably, when the T cells specific for a peptide according to thepresent invention are tested against the substituted peptides, thepeptide concentration at which the substituted peptides achieve half themaximal increase in lysis relative to background is no more than about 1mM, preferably no more than about 1 μM, more preferably no more thanabout 1 nM, and still more preferably no more than about 100 μM, andmost preferably no more than about 10 μM. It is also preferred that thesubstituted peptide be recognized by T cells from more than oneindividual, at least two, and more preferably three individuals.

In a particularly preferred embodiment of the invention the peptideconsists or consists essentially of an amino acid sequence according toSEQ ID NO: 1 to SEQ ID NO: 102.

“Consisting essentially of” shall mean that a peptide according to thepresent invention, in addition to the sequence according to any of SEQID NO: 1 to SEQ ID NO: 102 or a variant thereof contains additional N-and/or C-terminally located stretches of amino acids that are notnecessarily forming part of the peptide that functions as an epitope forMHC molecules.

Nevertheless, these stretches can be important to provide an efficientintroduction of the peptide according to the present invention into thecells. In one embodiment of the present invention, the peptide is partof a fusion protein which comprises, for example, the 80 N-terminalamino acids of the HLA-DR antigen-associated invariant chain (p33, inthe following “i”) as derived from the NCBI, GenBank Accession numberX00497. In other fusions, the peptides of the present invention can befused to an antibody as described herein, or a functional part thereof,in particular, into a sequence of an antibody, so as to be specificallytargeted by said antibody, or, for example, to or into an antibody thatis specific for dendritic cells as described herein.

In addition, the peptide or variant may be modified further to improvestability and/or binding to MHC molecules in order to elicit a strongerimmune response. Methods for such an optimization of a peptide sequenceare well known in the art and include, for example, the introduction ofreverse peptide bonds or non-peptide bonds.

In a reverse peptide bond, amino acid residues are not joined by peptide(—CO—NH—) linkages but the peptide bond is reversed. Such retro-inversopeptidomimetics may be made using methods known in the art, for examplesuch as those described by Meziere and colleagues (Meziere et al., 1997,incorporated herein by reference). This approach involves makingpseudopeptides containing changes involving the backbone, and not theorientation of side chains. They show that for MHC binding and T helpercell responses, these pseudopeptides are useful. Retro-inverse peptides,which contain NH—CO bonds instead of CO—NH peptide bonds, are much moreresistant to proteolysis (Meziere et al., 1997).

A non-peptide bond is, for example, —CH₂—NH, —CH₂S—, —CH₂CH₂—, —CH═CH—,—COCH₂—, —CH(OH)CH₂—, and —CH₂SO—. U.S. Pat. No. 4,897,445 provides amethod for the solid phase synthesis of non-peptide bonds (—CH₂—NH) inpolypeptide chains which involves polypeptides synthesized by standardprocedures and the non-peptide bond synthesized by reacting an aminoaldehyde and an amino acid in the presence of NaCNBH₃.

Peptides comprising the bonds described above may be synthesized withadditional chemical groups present at their amino and/or carboxytermini, to enhance the stability, bioavailability, and/or affinity ofthe peptides. For example, hydrophobic groups such as carbobenzoxyl,dansyl, or t-butyloxycarbonyl groups may be added to the peptides' aminotermini. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonylgroup may be placed at the peptides' amino termini. Additionally, thehydrophobic group, t-butyloxycarbonyl, or an amido group may be added tothe peptides' carboxy termini.

Further, the peptides of the invention may be synthesized to alter theirsteric configuration. For example, the D-isomer of one or more of theamino acid residues of the peptide may be used, rather than the usualL-isomer. Still further, at least one of the amino acid residues of thepeptides of the invention may be substituted by one of the well-knownnon-naturally occurring amino acid residues. Alterations such as thesemay serve to increase the stability, bioavailability and/or bindingaction of the peptides of the invention.

Similarly, a peptide or variant of the invention may be modifiedchemically by reacting specific amino acids either before or aftersynthesis of the peptide. Examples for such modifications are well knownin the art (Lundblad, 2004, which is incorporated herein by reference).Chemical modification of amino acids includes, but is not limited to,modification by acylation, amidination, pyridoxylation of lysine,reductive alkylation, trinitrobenzylation of amino groups with2,4,6-trinitrobenzene sulphonic acid (TNBS), amide modification ofcarboxyl groups and sulphydryl modification by performic acid oxidationof cysteine to cysteic acid, formation of mercurial derivatives,formation of mixed disulphides with other thiol compounds, reaction withmaleimide, carboxymethylation with iodoacetic acid or iodoacetamide andcarbamoylation with cyanate at alkaline pH, although without limitationthereto (Coligan et al., 1995, the contents of which are hereinincorporated by reference in their entirety).

Briefly, modification of e.g. arginyl residues in proteins is oftenbased on the reaction of vicinal dicarbonyl compounds such asphenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione to form anadduct. Another example is the reaction of methylglyoxal with arginylresidues. Cysteine can be modified without concomitant modification ofother nucleophilic sites such as lysine and histidine. As a result, alarge number of reagents are available for the modification of cysteine.The websites of companies such as Sigma-Aldrich (sigma-aldrich.com)provide information on specific reagents.

Selective reduction of disulfide bonds in proteins is also common.Disulfide bonds can be formed and oxidized during the heat treatment ofbiopharmaceuticals. Woodward's Reagent K may be used to modify specificglutamic acid residues. N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimidecan be used to form intra-molecular crosslinks between a lysine residueand a glutamic acid residue. For example, diethylpyrocarbonate is areagent for the modification of histidyl residues in proteins. Histidinecan also be modified using 4-hydroxy-2-nonenal. The reaction of lysineresidues and other α-amino groups is, for example, useful in binding ofpeptides to surfaces or the cross-linking of proteins/peptides. Lysineis the site of attachment of poly(ethylene)glycol and the major site ofmodification in the glycosylation of proteins. Methionine residues inproteins can be modified with e.g. iodoacetamide, bromoethylamine, andchloramine T.

Tetranitromethane and N-acetylimidazole can be used for the modificationof tyrosyl residues. Cross-linking via the formation of dityrosine canbe accomplished with hydrogen peroxide/copper ions.

Recent studies on the modification of tryptophan have usedN-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skatole).

Successful modification of therapeutic proteins and peptides with PEG isoften associated with an extension of circulatory half-life whilecross-linking proteins with glutaraldehyde, polyethylene glycoldiacrylate and formaldehyde is used for the preparation of hydrogels.Chemical modification of allergens for immunotherapy is often achievedby carbamylation with potassium cyanate.

Another embodiment of the present invention relates to a non-naturallyoccurring peptide wherein said peptide consists or consists essentiallyof an amino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 102and has been synthetically produced (e.g. synthesized) as apharmaceutically acceptable salt. Methods to synthetically producepeptides are well known in the art. The salts of the peptides accordingto the present invention differ substantially from the peptides in theirstate(s) in vivo, as the peptides generated in vivo are no salts. Thenon-natural salt form of the peptide mediates the solubility of thepeptide, in particular in the context of pharmaceutical compositionscomprising the peptides, e.g. the peptide vaccines as disclosed herein.A sufficient and at least substantial solubility of the peptide(s) isrequired in order to efficiently provide the peptides to the subject tobe treated. Preferably, the salts are pharmaceutically acceptable saltsof the peptides. These salts according to the invention include alkalineand earth alkaline salts such as salts of the Hofmeister seriescomprising as anions PO₄ ³, SO₄ ²⁻, CH₃COO⁻, Cl⁻, Br⁻, NO₃ ⁻, ClO₄ ⁻,I⁻, SCN⁻ and as cations NH₄ ⁺, Rb⁺, K⁺, Na⁺, Cs⁺, Li⁺, Zn²⁺, Mg²⁺, Ca²⁺,Mn²⁺, Cu²⁺ and Ba₂ ⁺. Particularly salts are selected from (NH₄)₃PO₄,(NH₄)₂HPO₄, (NH₄)H₂PO₄, (NH₄)₂SO₄, NH₄CH₃COO, NH₄Cl, NH₄Br, NH₄NO₃,NH₄ClO₄, NH₄I, NH₄SCN, Rb₃PO₄, Rb₂HPO₄, RbH₂PO₄, Rb₂SO₄, Rb₄CH₃COO,Rb₄Cl, Rb₄Br, Rb₄NO₃, Rb₄ClO₄, Rb₄I, Rb₄SCN, K₃PO₄, K₂HPO₄, KH₂PO₄,K₂SO₄, KCH₃COO, KCl, KBr, KNO₃, KClO₄, KI, KSCN, Na₃PO₄, Na₂HPO₄,NaH₂PO₄, Na₂SO₄, NaCH₃COO, NaCl, NaBr, NaNO₃, NaClO₄, NaI, NaSCN, ZnCl₂Cs₃PO₄, Cs₂HPO₄, CsH₂PO₄, Cs₂SO₄, CsCH₃COO, CsCl, CsBr, CsNO₃, CsClO₄,CsI, CsSCN, Li₃PO₄, Li₂HPO₄, LiH₂PO₄, Li₂SO₄, LiCH₃COO, LiCl, LiBr,LiNO₃, LiClO₄, Lil, LiSCN, Cu₂SO₄, Mg₃(PO₄)₂, Mg₂HPO₄, Mg(H₂PO₄)₂,Mg₂SO₄, Mg(CH₃COO)₂, MgCl₂, MgBr₂, Mg(NO₃)₂, Mg(ClO₄)₂, MgI₂, Mg(SCN)₂,MnCl₂, Ca₃(PO₄), Ca₂HPO₄, Ca(H₂PO₄)₂, CaSO₄, Ca(CH₃COO)₂, CaCl₂), CaBr₂,Ca(NO₃)₂, Ca(ClO₄)₂, CaI₂, Ca(SCN)₂, Ba₃(PO₄)₂, Ba₂HPO₄, Ba(H₂PO₄)₂,BaSO₄, Ba(CH₃COO)₂, BaCl₂, BaBr₂, Ba(NO₃)₂, Ba(ClO₄)₂, BaI₂, andBa(SCN)₂. Particularly preferred are NH acetate, MgCl₂, KH₂PO₄, Na₂SO₄,KCl, NaCl, and CaCl₂), such as, for example, the chloride or acetate(trifluoroacetate) salts (see e.g. Berge et al., 1977, the contents ofwhich are incorporated by reference in their entirety).

Generally, peptides and variants (at least those containing peptidelinkages between amino acid residues) may be synthesized by theFmoc-polyamide mode of solid-phase peptide synthesis as disclosed byLukas et al. (Lukas et al., 1981, the content of which is incorporatedby reference in its entirety) and by references as cited therein.Temporary N-amino group protection is afforded by the9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of thishighly base-labile protecting group is done using 20% piperidine in N,N-dimethylformamide. Side-chain functionalities may be protected astheir butyl ethers (in the case of serine, threonine and tyrosine),butyl esters (in the case of glutamic acid and aspartic acid),butyloxycarbonyl derivative (in the case of lysine and histidine),trityl derivative (in the case of cysteine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). Where glutamine or asparagine are C-terminal residues, use ismade of the 4,4′-dimethoxybenzhydryl group for protection of the sidechain amido functionalities. The solid-phase support is based on apolydimethyl-acrylamide polymer constituted from the three monomersdimethylacrylamide (backbone-monomer), bisacryloylethylene diamine(cross linker) and acryloylsarcosine methyl ester (functionalizingagent). The peptide-to-resin cleavable linked agent used is theacid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All aminoacid derivatives are added as their preformed symmetrical anhydridederivatives with the exception of asparagine and glutamine, which areadded using a reversed N,N-dicyclohexyl-carbodiimide/ihydroxybenzotriazole mediated coupling procedure. All coupling anddeprotection reactions are monitored using ninhydrin, trinitrobenzenesulphonic acid or isotin test procedures. Upon completion of synthesis,peptides are cleaved from the resin support with concomitant removal ofside-chain protecting groups by treatment with 95% trifluoroacetic acidcontaining a 50% scavenger mix. Scavengers commonly used includeethanedithiol, phenol, anisole and water, the exact choice depending onthe constituent amino acids of the peptide being synthesized. Also, acombination of solid phase and solution phase methodologies for thesynthesis of peptides is possible (see for example Bruckdorfer et al.,2004 and the references as cited therein, the content of which isincorporated by reference in its entirety).

Trifluoroacetic acid is removed by evaporation in vacuo, with subsequenttrituration with diethyl ether affording the crude peptide. Anyscavengers present are removed by a simple extraction procedure which onlyophilization of the aqueous phase affords the crude peptide free ofscavengers. Reagents for peptide synthesis are generally available frome.g. Calbiochem-Novabiochem (Nottingham, UK).

Purification may be performed by anyone, or a combination of techniquessuch as re-crystallization, size exclusion chromatography, ion-exchangechromatography, hydrophobic interaction chromatography and (usually)reverse-phase high performance liquid chromatography using e.g.acetonitrile/water gradient separation.

Analysis of peptides may be carried out using thin layer chromatography,electrophoresis, in particular capillary electrophoresis, solid phaseextraction (CSPE), reverse-phase high performance liquid chromatography,amino-acid analysis after acid hydrolysis and by fast atom bombardment(FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF massspectrometric analysis.

In order to select over-presented peptides, a presentation profile iscalculated showing the median sample presentation as well as replicatevariation. The profile juxtaposes samples of the tumor entity ofinterest to a baseline of normal tissue samples. Each of these profilescan then be consolidated into an over-presentation score by calculatingthe p-value of a Linear Mixed-Effects Model (Pinheiro et al., 2015)adjusting for multiple testing by False Discovery Rate (Benjamini andHochberg, 1995, the content of which is incorporated by reference in itsentirety).

For the identification and relative quantitation of HLA ligands by massspectrometry, HLA molecules from shock-frozen tissue samples werepurified and HLA associated peptides were isolated. The isolatedpeptides were separated, and sequences were identified by onlinenano-electrospray-ionization (nanoESI) liquid chromatography-massspectrometry (LC-MS) experiments. The resulting peptide sequences wereverified by comparison of the fragmentation pattern of naturaltumor-associated peptides (TUMAPs) recorded from acute myeloid leukemia,breast cancer, cholangiocellular carcinoma, chronic lymphocyticleukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastriccancer, gastro-esophageal junction cancer, hepatocellular carcinoma,head and neck squamous cell carcinoma, melanoma, non-Hodgkin lymphoma,non-small cell lung cancer, ovarian cancer, esophageal cancer,pancreatic cancer, prostate cancer, renal cell carcinoma, small celllung cancer, urinary bladder carcinoma, and uterine endometrial cancersamples (N>750 samples) with the fragmentation patterns of correspondingsynthetic reference peptides of identical sequences. Since the peptideswere directly identified as ligands of HLA molecules of primary tumors,these results provide direct evidence for the natural processing andpresentation of the identified peptides on primary cancer tissueobtained from acute myeloid leukemia, breast cancer, cholangiocellularcarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladdercancer, glioblastoma, gastric cancer, gastro-esophageal junction cancer,hepatocellular carcinoma, head and neck squamous cell carcinoma,melanoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovariancancer, esophageal cancer, pancreatic cancer, prostate cancer, renalcell carcinoma, small cell lung cancer, urinary bladder carcinoma, anduterine endometrial cancer patients (cf. Example 1, FIGS. 3A-3D).

The discovery pipeline XPRESIDENT® v2.1 allows the identification andselection of relevant over-presented peptides which are potentialtargets for immunotherapy based on direct relative quantitation of HLArestricted peptide levels on cancer tissues in comparison to severaldifferent non-cancerous tissues and organs. See e.g. U.S. PatentApplication Publication No. 2013/0096016 the contents of which areincorporated by reference in their entirety. This was achieved by thedevelopment of label-free differential quantitation using the acquiredLC-MS data processed by a proprietary data analysis pipeline, combiningalgorithms for sequence identification, spectral clustering, ioncounting, retention time alignment, charge state deconvolution andnormalization.

Additional sequence information from public resources (Olexiouk et al.,2016; Subramanian et al., 2011, the contents which are incorporated byreference in their entirety) were integrated into the XPRESIDENT®discovery pipeline to enable the identification of TUMAPs fromnon-canonical origin. Presentation levels including error estimates foreach peptide and sample were established. Peptides exclusively presentedon tumor tissue and peptides over-presented in tumor versusnon-cancerous tissues and organs have been identified.

HLA-peptide complexes from acute myeloid leukemia, breast cancer,cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectalcancer, gallbladder cancer, glioblastoma, gastric cancer,gastro-esophageal junction cancer, hepatocellular carcinoma, head andneck squamous cell carcinoma, melanoma, non-Hodgkin lymphoma, non-smallcell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, small cell lung cancer, urinarybladder carcinoma, and uterine endometrial cancer tissue samples werepurified and HLA associated peptides were isolated and analyzed by LC-MS(see Example 1). All TUMAPs contained in the present application wereidentified with this approach on acute myeloid leukemia, breast cancer,cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectalcancer, gallbladder cancer, glioblastoma, gastric cancer,gastro-esophageal junction cancer, hepatocellular carcinoma, head andneck squamous cell carcinoma, melanoma, non-Hodgkin lymphoma, non-smallcell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, small cell lung cancer, urinarybladder carcinoma, and uterine endometrial cancer samples confirmingtheir presentation on acute myeloid leukemia, breast cancer,cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectalcancer, gallbladder cancer, glioblastoma, gastric cancer,gastro-esophageal junction cancer, hepatocellular carcinoma, head andneck squamous cell carcinoma, melanoma, non-Hodgkin lymphoma, non-smallcell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, small cell lung cancer, urinarybladder carcinoma, uterine endometrial cancer, and combinations thereof.

TUMAPs identified on multiple acute myeloid leukemia, breast cancer,cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectalcancer, gallbladder cancer, glioblastoma, gastric cancer,gastro-esophageal junction cancer, hepatocellular carcinoma, head andneck squamous cell carcinoma, melanoma, non-Hodgkin lymphoma, non-smallcell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, small cell lung cancer, urinarybladder carcinoma, and uterine endometrial cancer and normal tissueswere quantified using ion-counting of label-free LC-MS data. The methodassumes that LC-MS signal areas of a peptide correlate with itsabundance in the sample. All quantitative signals of a peptide invarious LC-MS experiments were normalized based on central tendency,averaged per sample and merged into a bar plot, called presentationprofile. The presentation profile consolidates different analysismethods like protein database search, spectral clustering, charge statedeconvolution (decharging) and retention time alignment andnormalization.

Furthermore, the discovery pipeline XPRESIDENT® allows the directabsolute quantitation of MHC-, preferably HLA restricted, peptide levelson cancer or other infected tissues. Briefly, the total cell count wascalculated from the total DNA content of the analyzed tissue sample. Thetotal peptide amount for a TUMAP in a tissue sample was measured by nanoLC-MS/MS as the ratio of the natural TUMAP and a known amount of anisotope-labelled version of the TUMAP, the so-called internal standard.The efficiency of TUMAP isolation was determined by spiking peptide-MHCcomplexes of all selected TUMAPs into the tissue lysate at the earliestpossible point of the TUMAP isolation procedure and their detection bynano LC-MS/MS following completion of the peptide isolation procedure.The total cell count and the amount of total peptide were calculatedfrom triplicate measurements per tissue sample. The peptide specificisolation efficiencies were calculated as an average from 9 spikeexperiments each measured as a triplicate (see Example 4 and Table 12).

Besides over-presentation of the peptide, mRNA expression of theunderlying gene was tested. mRNA data were obtained via RNASeq analysesof normal tissues and cancer tissues (see Example 2, FIGS. 4A-4D). Anadditional source of normal tissue data was a database of publiclyavailable RNA expression data from around 3000 normal tissue samples(Lonsdale, 2013, the content of which is incorporated by reference inits entirety). Peptides which are derived from proteins whose codingmRNA is highly expressed in cancer tissue, but very low or absent invital normal tissues, were preferably included in the present invention.

The present invention provides peptides that are useful in treatingcancers/tumors, preferably acute myeloid leukemia, breast cancer,cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectalcancer, gallbladder cancer, glioblastoma, gastric cancer,gastro-esophageal junction cancer, hepatocellular carcinoma, head andneck squamous cell carcinoma, melanoma, non-Hodgkin lymphoma, non-smallcell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, small cell lung cancer, urinarybladder carcinoma, and uterine endometrial cancer that over- orexclusively present the peptides of the invention. These peptides wereshown by MS to be naturally presented by HLA molecules on primary humanacute myeloid leukemia, breast cancer, cholangiocellular carcinoma,chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, gastro-esophageal junction cancer,hepatocellular carcinoma, head and neck squamous cell carcinoma,melanoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovariancancer, esophageal cancer, pancreatic cancer, prostate cancer, renalcell carcinoma, small cell lung cancer, urinary bladder carcinoma, anduterine endometrial cancer samples.

Many of the source genes/proteins (also designated “full-lengthproteins” or “underlying proteins”) from which the peptides are derivedwere shown to be highly overexpressed in cancer compared with normaltissues—“normal tissues” in relation to this invention shall mean eitherhealthy blood, brain, heart, liver, lung, adipose tissue, adrenal gland,bile duct, bladder, bone, bone marrow, esophagus, eye, gallbladder,head&neck, large intestine, small intestine, kidney, lymph node, centralnerve, peripheral nerve, pancreas, parathyroid gland, peritoneum,pituitary, pleura, skeletal muscle, skin, spinal cord, spleen, stomach,thyroid, trachea, and ureter cells or other normal tissue cells such asbreast, ovary, placenta, prostate, testis, thymus and uterus,demonstrating a high degree of tumor association of the source genes(see Example 2). Moreover, the peptides themselves are stronglyover-presented on tumor tissue—“tumor tissue” in relation to thisinvention shall mean a sample from a patient suffering from acutemyeloid leukemia, breast cancer, cholangiocellular carcinoma, chroniclymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, gastro-esophageal junction cancer,hepatocellular carcinoma, head and neck squamous cell carcinoma,melanoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovariancancer, esophageal cancer, pancreatic cancer, prostate cancer, renalcell carcinoma, small cell lung cancer, urinary bladder carcinoma, anduterine endometrial cancer, but not on normal tissues (see Example 1).

HLA bound peptides can be recognized by the immune system, specificallyT lymphocytes. T cells can destroy the cells presenting the recognizedHLA-peptide complex, e.g. acute myeloid leukemia, breast cancer,cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectalcancer, gallbladder cancer, glioblastoma, gastric cancer,gastro-esophageal junction cancer, hepatocellular carcinoma, head andneck squamous cell carcinoma, melanoma, non-Hodgkin lymphoma, non-smallcell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, small cell lung cancer, urinarybladder carcinoma, and uterine endometrial cancer cells presenting thederived peptides.

The peptides of the present invention over-presented in cancer tissuecompared to normal tissues and thus can be used for the production ofantibodies and/or TCRs, such as soluble TCRs, according to the presentinvention (see Table 9). Furthermore, the peptides when complexed withthe respective MHC can be used for the production of antibodies and/orTCRs, in particular soluble TCRs, according to the present invention, aswell. Respective methods are well known to the person of skill and canbe found in the respective literature as well (see also below). Thus,the peptides of the present invention are useful for generating animmune response in a patient by which tumor cells can be destroyed. Animmune response in a patient can be induced by direct administration ofthe described peptides or suitable precursor substances (e.g. elongatedpeptides, proteins, or nucleic acids encoding these peptides) to thepatient, ideally in combination with an agent enhancing theimmunogenicity (i.e., an adjuvant). The immune response originating fromsuch a therapeutic vaccination can be expected to be highly specificagainst tumor cells because the target peptides of the present inventionare not presented on normal tissues in comparable copy numbers,preventing the risk of undesired autoimmune reactions against normalcells in the patient.

The present description further relates to TCRs comprising an alphachain and a beta chain (“alpha/beta TCRs”). Also provided are peptidesaccording to the invention capable of binding to TCRs and antibodieswhen presented by an MHC molecule.

The present description also relates to fragments of the TCRs accordingto the invention that are capable of binding to a peptide antigenaccording to the present invention when presented by an HLA molecule.The term particularly relates to soluble TCR fragments, for example TCRsmissing the transmembrane parts and/or constant regions, single chainTCRs, and fusions thereof for example to Immunoglobulin.

The present description also relates to nucleic acids, vectors and hostcells expressing TCRs and peptides of the present description; andmethods of using the same.

The term “T cell receptor” (abbreviated TCR) refers to a heterodimericmolecule comprising an alpha polypeptide chain (alpha chain) and a betapolypeptide chain (beta chain), wherein the heterodimeric receptor iscapable of binding to a peptide antigen presented by an HLA molecule.The term also includes so-called gamma/delta TCRs.

In one embodiment the description provides a method of producing a TCRas described herein, the method comprising culturing a host cell capableof expressing the TCR under conditions suitable to promote expression ofthe TCR.

The description in another aspect relates to methods according to thedescription, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell or the antigen is loadedonto class I or II MHC tetramers by tetramerizing the antigen/class I orII MHC complex monomers.

The alpha and beta chains of alpha/beta TCR's, and the gamma and deltachains of gamma/delta TCRs, are generally regarded as each having two“domains”, namely variable and constant domains. The variable domainconsists of a concatenation of the variable region (V) and the joiningregion (J). The variable domain may also include a leader region (L).Beta and delta chains may also include a diversity region (D). The alphaand beta constant domains may also include C-terminal transmembrane (TM)domains that anchor the alpha and beta chains to the cell membrane.

With respect to gamma/delta TCRs, the term “TCR gamma variable domain”as used herein refers to the concatenation of the TCR gamma V (TRGV)region without leader region (L), and the TCR gamma J (TRGJ) region, andthe term TCR gamma constant domain refers to the extracellular TRGCregion, or to a C-terminal truncated TRGC sequence. Likewise, the term“TCR delta variable domain” refers to the concatenation of the TCR deltaV (TRDV) region without leader region (L) and the TCR delta D/J(TRDD/TRDJ) region, and the term “TCR delta constant domain” refers tothe extracellular TRDC region, or to a C-terminal truncated TRDCsequence.

Alpha/beta heterodimeric TCRs of the present description may have anintroduced disulfide bond between their constant domains. Preferred TCRsof this type include those which have a TRAC constant domain sequenceand a TRBC1 or TRBC2 constant domain sequence except that Thr 48 of TRACand Ser 57 of TRBC1 or TRBC2 are replaced by cysteine residues, the saidcysteines forming a disulfide bond between the TRAC constant domainsequence and the TRBC1 or TRBC2 constant domain sequence of the TCR.

With or without the introduced inter-chain bond mentioned above,alpha/beta heterodimeric TCRs of the present description may have a TRACconstant domain sequence and a TRBC1 or TRBC2 constant domain sequence,and the TRAC constant domain sequence and the TRBC1 or TRBC2 constantdomain sequence of the TCR may be linked by the native disulfide bondbetween Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2.

TCRs of the present description may comprise a detectable label selectedfrom the group consisting of a radionuclide, a fluorophore and biotin.TCRs of the present description may be conjugated to a therapeuticallyactive agent, such as a radionuclide, a chemotherapeutic agent, or atoxin.

Detectable Labels

Detectable labels may be for example fluorescent dyes, enzymes,substrates, bioluminescent materials, radioactive materials, andchemiluminescent labels. Exemplary enzymes labels include, but are notlimited to, horseradish peroxidase, acetylcholinesterase, alkalinephosphatase, b-galactosidase and luciferase. Exemplary fluorophores(fluorescent materials) include, but are not limited to, rhodamine,fluorescein, fluorescein isothiocyanate, umbelliferone,dichlorotriazinylamine, phycoerythrin and dansyl chloride. Exemplarychemiluminescent labels include, but are not limited to, luminol.Exemplary bioluminescent materials include, but are not limited to,luciferin and aequorin. Exemplary radioactive materials include, but arenot limited to, bismuth-213 (²¹³Bs), carbon-14 (¹⁴C), carbon-11 (¹¹C),chlorine-18 (¹⁸Cl), chromium-51 (⁵¹Cr), cobalt-57 (⁵⁷Co), cobalt-60(⁶⁰Co), copper-64 (⁶⁴Cu), copper-67 (⁶⁷Cu), dysprosium-165 (¹⁶⁵Dy),erbium-169 (¹⁶⁹Er), fluorine-18 (¹⁸F), gallium-67 (⁶⁷Ga), gallium-68(⁶⁸Ga), germanium-68 (⁶⁸Ge), holmium-166 (¹⁶⁶Ho), indium-111 (¹¹¹1n),iodine-123 (¹²³1), iodine-124 (¹²⁴1), iodine-125 (¹²⁵I), iodine-131(¹³¹I), iridium-192 (¹⁹²1r), iron-59 (⁵⁹Fe), krypton-81 (⁸¹Kr), lead-212(²¹²Pb), lutetium-177 (¹⁷⁷Lu), molybdenum-99 (⁹⁹Mo), nitrogen-13 (¹³N),oxygen-15 (¹⁵O), palladium-103 (¹⁰³Pd), phosphorus-32 (³²P),potassium-42 (⁴²K), rhenium-186 (¹⁸⁶Re), rhenium-188 (¹⁸⁸Re),rubidium-81 (⁸¹Rb), rubidium-82 (⁸²Rb), samarium-153 (¹⁵3Sm),selenium-75 (⁷⁵Se), sodium-24 (²⁴Na), strontium-82 (⁸²Sr), strontium-89(⁸⁹Sr), sulfur 35 (³⁵S), technetium-99m (⁹⁹Tc), thallium-201 (²⁰¹Tl),tritium (³H), xenon-133 (¹³³Xe), ytterbium-169 (¹⁶⁹Yb), ytterbium-177(¹⁷⁷Yb), and yttrium-90 (⁹⁰Y).

Radionuclides

Radionuclides emit alpha or beta particles (e.g. radioimmunoconjugates).Such radioactive isotopes include, but are not limited to, beta-emitterssuch as phosphorus-32 (³²P), scandium-47 (⁴⁷Sc), copper-67 (⁶⁷Cu),gallium-67 (⁶⁷Ga), yttrium-88 (⁸⁸Y), yttrium-90 (⁹⁰Y), iodine-125(¹²⁵I), iodine-131 (¹³¹I), samarium-153 (¹⁵³Sm), lutetium-177 (¹⁷⁷Lu),rhenium-186 (¹⁸⁶Re), rhenium-188 (¹⁸⁸Re), and alpha-emitters such asastatine-211 (²¹¹At), lead-212 (²¹²Pb), bismuth-212 (²¹²Bi), bismuth-213(²¹³Bi) or actinium-225 (²²⁵Ac).

Toxins

Toxins include, but are not limited to, methotrexate, aminopterin,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil dacarbazine;alkylating agents such as mechlorethamine, thiotepa chlorambucil,melphalan, carmustine (BSNU), mitomycin C, lomustine (CCNU),1-methylnitrosourea, cyclophosphamide, mechlorethamine, busulfan,dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamineplatinum (II) (DDP) cisplatin and carboplatin (paraplatin);anthracyclines include daunorubicin (formerly daunomycin), doxorubicin(adriamycin), detorubicin, caminomycin, idarubicin, epirubicin,mitoxantrone and bisantrene; antibiotics include dactinomycin(actinomycin D), bleomycin, calicheamicin, mithramycin, and anthramycin(AMC); and antimytotic agents such as the vinca alkaloids, vincristineand vinblastine. Other cytotoxic agents include paclitaxel (TAXOL®),ricin, pseudomonas exotoxin, gemcitabine, cytochalasin B, gramicidin D,ethidium bromide, emetine, etoposide, tenoposide, colchicin, dihydroxyanthracin dione, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, puromycin, procarbazine,hydroxyurea, asparaginase, corticosteroids, mytotane (O,P′-(DDD)),interferons, and mixtures of these cytotoxic agents

Therapeutic agents include, but are not limited to, carboplatin,cisplatin, paclitaxel, gemcitabine, calicheamicin, doxorubicin,5-fluorouracil, mitomycin C, actinomycin D, cyclophosphamide,vincristine, bleomycin, VEGF antagonists, EGFR antagonists, platins,taxols, irinotecan, 5-fluorouracil, gemcytabine, leucovorine, steroids,cyclophosphamide, melphalan, vinca alkaloids, mustines, tyrosine kinaseinhibitors, radiotherapy, sex hormone antagonists, selective androgenreceptor modulators, selective estrogen receptor modulators, PDGFantagonists, TNF antagonists, IL-1 antagonists, interleukins (e.g. IL-12or IL-2), IL-12R antagonists, Toxin conjugated monoclonal antibodies,tumor antigen specific monoclonal antibodies, Erbitux®, Avastin®,Pertuzumab, anti-CD20 antibodies, Rituxan, RTM, ocrelizumab, ofatumumab,DXL625, Herceptin®, or any combination thereof. Toxic enzymes fromplants and bacteria such as ricin, diphtheria toxin and Pseudomonastoxin may be used to generate cell type specific killing reagents (Youleet al., 1980, Gilliland et al., 1980, Krolick et al., 1980). Othercytotoxic agents include cytotoxic ribonucleases (See U.S. Pat. No.6,653,104, the contents of each reference which are incorporated byreference in their entireties).

In an embodiment, a TCR of the present description having at least onemutation in the alpha chain and/or having at least one mutation in thebeta chain has modified glycosylation compared to the unmutated TCR.

In an embodiment, a TCR comprising at least one mutation in the TCRalpha chain and/or TCR beta chain has a binding affinity for, and/or abinding half-life for, a peptide-HLA molecule complex, which is at leastdouble that of a TCR comprising the unmutated TCR alpha chain and/orunmutated TCR beta chain. Affinity-enhancement of tumor specific TCRs,and its exploitation, relies on the existence of a window for optimalTCR affinities. The existence of such a window is based on observationsthat TCRs specific for e.g. HLA-A*02-restricted pathogens have K_(d)values that are generally about 10-fold lower when compared to TCRsspecific for e.g. HLA-A*02-restricted tumor-associated self-antigens. Itis now known, although tumor antigens have the potential to beimmunogenic, because tumors arise from the individual's own cells onlymutated proteins or proteins with altered translational processing willbe seen as foreign by the immune system. Antigens that are upregulatedor overexpressed (so called self-antigens) will not necessarily induce afunctional immune response against the tumor: T cells expressing TCRsthat are highly reactive to these antigens will have been negativelyselected within the thymus in a process known as central tolerance,meaning that only T cells with low-affinity TCRs for self-antigensremain. Therefore, affinity of TCRs or variants of the presentdescription to peptides can be enhanced by methods well known in theart.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising incubating PBMCs from HLA-A*02-negative healthy donors withHLA-A*02-peptide monomers, incubating the PBMCs withtetramer-phycoerythrin (PE) and isolating the high avidity T cells byfluorescence activated cell sorting (FACS) Calibur analysis.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising obtaining a transgenic mouse with the entire human TCRαβ geneloci (1.1 and 0.7 Mb), whose T cells express a diverse human TCRrepertoire that compensates for mouse TCR deficiency, immunizing themouse with a peptide, incubating PBMCs obtained from the transgenic micewith tetramer-phycoerythrin (PE), and isolating the high avidity T cellsby fluorescence activated cell sorting (FACS) Calibur analysis.

In one aspect, to obtain T cells expressing TCRs of the presentdescription, nucleic acids encoding TCR-alpha and/or TCR-beta chains ofthe present description are cloned into expression vectors, such asgamma retrovirus or lentivirus. The recombinant viruses are generatedand then tested for functionality, such as antigen specificity andfunctional avidity. An aliquot of the final product is then used totransduce the target T cell population (generally purified from patientPBMCs), which is expanded before infusion into the patient.

In another aspect, to obtain T cells expressing TCRs of the presentdescription, TCR RNAs are synthesized by techniques known in the arte.g. in vitro transcription systems. The in vitro synthesized TCR RNAsare then introduced into primary CD8-positive T cells obtained fromhealthy donors by electroporation to re-express tumor specific TCR-alphaand/or TCR-beta chains.

To increase the expression, nucleic acids encoding TCRs of the presentdescription may be operably linked to strong promoters, such asretroviral long terminal repeats (LTRs), cytomegalovirus (CMV), murinestem cell virus (MSCV) U3, phosphoglycerate kinase (PGK), β-actin,ubiquitin, and a simian virus 40 (SV40)/CD43 composite promoter,elongation factor (EF)-1a and the spleen focus-forming virus (SFFV)promoter. In a preferred embodiment, the promoter is heterologous to thenucleic acid being expressed.

In addition to strong promoters, TCR expression cassettes of the presentdescription may contain additional elements that can enhance transgeneexpression, including a central polypurine tract (cPPT), which promotesthe nuclear translocation of lentiviral constructs (Follenzi et al.,2000), and the woodchuck hepatitis virus post-transcriptional regulatoryelement (wPRE), which increases the level of transgene expression byincreasing RNA stability (Zufferey et al., 1999, the contents of whichare incorporated by reference in their entirety).

The alpha and beta chains of a TCR of the present invention may beencoded by nucleic acids located in separate vectors or may be encodedby polynucleotides located in the same vector.

Achieving high level TCR surface expression requires that both theTCR-alpha and TCR-beta chains of the introduced TCR be transcribed athigh levels. To do so, the TCR-alpha and TCR-beta chains of the presentdescription may be cloned into bi-cistronic constructs in a singlevector, which has been shown to be capable of overcoming this obstacle.The use of a viral internal ribosomal entry site (IRES) between theTCR-alpha and TCR-beta chains results in the coordinated expression ofboth chains, because the TCR-alpha and TCR-beta chains are generatedfrom a single transcript that is broken into two proteins duringtranslation, ensuring that an equal molar ratio of TCR-alpha andTCR-beta chains are produced (Schmitt et al., 2009, the contents ofwhich are incorporated by reference in their entirety).

Nucleic acids encoding TCRs of the present description may be codonoptimized to increase expression from a host cell. Redundancy in thegenetic code allows some amino acids to be encoded by more than onecodon, but certain codons are less “optimal” than others because of therelative availability of matching tRNAs as well as other factors(Gustafsson et al., 2004). Modifying the TCR-alpha and TCR-beta genesequences such that each amino acid is encoded by the optimal codon formammalian gene expression, as well as eliminating mRNA instabilitymotifs or cryptic splice sites, has been shown to significantly enhanceTCR-alpha and TCR-beta gene expression (Scholten et al., 2006, thecontents of these references are herein incorporated by reference intheir entirety).

Furthermore, mispairing between the introduced and endogenous TCR chainsmay result in the acquisition of specificities that pose a significantrisk for autoimmunity. For example, the formation of mixed TCR dimersmay reduce the number of CD3 molecules available to form properly pairedTCR complexes, and therefore can significantly decrease the functionalavidity of the cells expressing the introduced TCR (Kuball et al., 2007,the contents of which are incorporated by reference in their entirety).

To reduce mispairing, the C-terminus domain of the introduced TCR chainsof the present description may be modified in order to promoteinterchain affinity, while decreasing the ability of the introducedchains to pair with the endogenous TCR. These strategies may includereplacing the human TCR-alpha and TCR-beta C-terminus domains with theirmurine counterparts (murinized C-terminus domain); generating a secondinterchain disulfide bond in the C-terminus domain by introducing asecond cysteine residue into both the TCR-alpha and TCR-beta chains ofthe introduced TCR (cysteine modification); swapping interactingresidues in the TCR-alpha and TCR-beta chain C-terminus domains(“knob-in-hole”); and fusing the variable domains of the TCR-alpha andTCR-beta chains directly to CD3ζ (CD3ζ (fusion) (Schmitt et al., 2009).

In an embodiment, a host cell is engineered to express a TCR of thepresent description. In preferred embodiments, the host cell is a humanT cell or T cell progenitor. In some embodiments the T cell or T cellprogenitor is obtained from a cancer patient. In other embodiments the Tcell or T cell progenitor is obtained from a healthy donor. Host cellsof the present description can be allogeneic or autologous with respectto a patient to be treated. In one embodiment, the host is a gamma/deltaT cell transformed to express an alpha/beta TCR.

A “pharmaceutical composition” is a composition suitable foradministration to a human being in a medical setting. Preferably, apharmaceutical composition is sterile and produced according to GMPguidelines.

The pharmaceutical compositions comprise the peptides either in the freeform or in the form of a pharmaceutically acceptable salt (see alsoabove). As used herein, “a pharmaceutically acceptable salt” refers to aderivative of the disclosed peptides wherein the peptide is modified bymaking acid or base salts of the agent. For example, acid salts areprepared from the free base (typically wherein the neutral form of thedrug has a neutral —NH₂ group) involving reaction with a suitable acid.Suitable acids for preparing acid salts include both organic acids e.g.acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,malic acid, malonic acid, succinic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methane sulfonic acid, ethane sulfonic acid, p-toluenesulfonic acid,salicylic acid, and the like, as well as inorganic acids, e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acidphosphoric acid and the like. Conversely, preparation of basic salts ofacid moieties which may be present on a peptide are prepared using apharmaceutically acceptable base such as sodium hydroxide, potassiumhydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or thelike.

In an especially preferred embodiment, the pharmaceutical compositionscomprise the peptides as salts of acetic acid (acetates), trifluoroacetates or hydrochloric acid (chlorides).

Preferably, the medicament of the present invention is animmunotherapeutic such as a vaccine. It may be administered directlyinto the patient, into the affected organ or systemically intravenous(i.v.), sub-cutaneous (s.c.), intradermal (i.d.), intraperitoneal(i.p.), intramuscular (i.m.) or applied ex vivo to cells derived fromthe patient or a human cell line which are subsequently administered tothe patient or used in vitro to select a subpopulation of immune cellsderived from the patient, which are then re-administered to the patient.If the nucleic acid is administered to cells in vitro, it may be usefulfor the cells to be transfected so as to co-express immune-stimulatingcytokines, such as Interleukin-2. The peptide may be substantially pureor combined with an immune-stimulating adjuvant (see below) or used incombination with immune-stimulatory cytokines, or be administered with asuitable delivery system, for example liposomes. The peptide may also beconjugated to a suitable carrier such as keyhole limpet hemocyanin (KLH)or mannan (see WO 95/18145 and Longenecker et al., 1993, the contentsboth of which are incorporated by reference in their entirety). Thepeptide may also be tagged, may be a fusion protein, or may be a hybridmolecule. The peptides whose sequences are given in the presentinvention are expected to stimulate CD4 or CD8 T cells. However,stimulation of CD8 T cells is more efficient in the presence of helpprovided by CD4 T helper cells. Thus, for MHC class I epitopes thatstimulate CD8 T cells the fusion partner or sections of a hybridmolecule suitably provide epitopes which stimulate CD4-positive T cells.CD4- and CD8-stimulating epitopes are well known in the art and includethose identified in the present invention.

In one aspect, the vaccine comprises at least one peptide having theamino acid sequence set forth SEQ ID NO: 1 to SEQ ID NO: 102, and atleast one additional peptide, preferably two to 50, more preferably twoto 25, even more preferably two to 20 and most preferably two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, fifteen, sixteen, seventeen or eighteen peptides. Thepeptide(s) may be derived from one or more specific TAAs and may bind toMHC class I molecules.

A further aspect of the invention provides a nucleic acid (for example apolynucleotide) encoding a peptide or peptide variant of the invention.The polynucleotide may be, for example, DNA, cDNA, PNA, RNA orcombinations thereof, either single- and/or double-stranded, or nativeor stabilized forms of polynucleotides, such as, for example,polynucleotides with a phosphorothioate backbone and it may or may notcontain introns so long as it codes for the peptide. Of course, onlypeptides that contain naturally occurring amino acid residues joined bynaturally occurring peptide bonds are encodable by a polynucleotide. Astill further aspect of the invention provides an expression vectorcapable of expressing a polypeptide according to the invention.

A variety of methods have been developed to link polynucleotides,especially DNA, to vectors for example via complementary cohesivetermini. For instance, complementary homopolymer tracts can be added tothe DNA segment to be inserted to the vector DNA. The vector and DNAsegment are then joined by hydrogen bonding between the complementaryhomopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. Syntheticlinkers containing a variety of restriction endonuclease sites arecommercially available from a number of sources including InternationalBiotechnologies Inc. New Haven, CN, USA.

A desirable method of modifying the DNA encoding the polypeptide of theinvention employs the polymerase chain reaction as disclosed by Saiki etal. (Saiki et al., 1988, the contents of which are incorporated byreference in their entirety). This method may be used for introducingthe DNA into a suitable vector, for example by engineering in suitablerestriction sites, or it may be used to modify the DNA in other usefulways as is known in the art. If viral vectors are used, pox- oradenovirus vectors are preferred.

The DNA (or in the case of retroviral vectors, RNA) may then beexpressed in a suitable host to produce a polypeptide comprising thepeptide or variant of the invention. Thus, the DNA encoding the peptideor variant of the invention may be used in accordance with knowntechniques, appropriately modified in view of the teachings containedherein, to construct an expression vector, which is then used totransform an appropriate host cell for the expression and production ofthe polypeptide of the invention. Such techniques include thosedisclosed for example in U.S. Pat. Nos. 4,440,859, 4,530,901, 4,582,800,4,677,063, 4,678,751, 4,704,362, 4,710,463, 4,757,006, 4,766,075, and4,810,648, the contents each of which are incorporated by reference intheir entirety.

The DNA (or in the case of retroviral vectors, RNA) encoding thepolypeptide constituting the compound of the invention may be joined toa wide variety of other DNA sequences for introduction into anappropriate host. The companion DNA will depend upon the nature of thehost, the manner of the introduction of the DNA into the host, andwhether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognized bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques. Generally, not all of the hosts will be transformedby the vector. Therefore, it will be necessary to select for transformedhost cells. One selection technique involves incorporating a DNAsequence into the expression vector, with any necessary controlelements, that codes for a selectable trait in the transformed cell,such as antibiotic resistance.

Alternatively, the gene for such selectable trait can be on anothervector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the polypeptide, which canthen be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae), filamentous fungi (for example Aspergillus spec.), plantcells, animal cells and insect cells. Preferably, the system can bemammalian cells such as CHO cells available from the ATCC Cell BiologyCollection.

A typical mammalian cell vector plasmid for constitutive expressioncomprises the CMV or SV40 promoter with a suitable poly A tail and aresistance marker, such as neomycin. One example is pSVL available fromPharmacia, Piscataway, NJ, USA. An example of an inducible mammalianexpression vector is pMSG, also available from Pharmacia. Useful yeastplasmid vectors are pRS403-406 and pRS413-416 and are generallyavailable from Stratagene Cloning Systems, La Jolla, CA 92037, USA.Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integratingplasmids (Ylps) and incorporate the yeast selectable markers HIS3, TRP1,LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).CMV promoter-based vectors (for example from Sigma-Aldrich) providetransient or stable expression, cytoplasmic expression or secretion, andN-terminal or C-terminal tagging in various combinations of FLAG,3×FLAG, c-myc or MAT. These fusion proteins allow for detection,purification and analysis of recombinant protein. Dual-tagged fusionsprovide flexibility in detection.

The strong human cytomegalovirus (CMV) promoter regulatory region drivesconstitutive protein expression levels as high as 1 mg/L in COS cells.For less potent cell lines, protein levels are typically ˜0.1 mg/L. Thepresence of the SV40 replication origin will result in high levels ofDNA replication in SV40 replication permissive COS cells. CMV vectors,for example, can contain the pMB1 (derivative of pBR322) origin forreplication in bacterial cells, the b-lactamase gene for ampicillinresistance selection in bacteria, hGH polyA, and the f1 origin. Vectorscontaining the pre-pro-trypsin leader (PPT) sequence can direct thesecretion of FLAG fusion proteins into the culture medium forpurification using ANTI-FLAG antibodies, resins, and plates. Othervectors and expression systems are well known in the art for use with avariety of host cells.

In another embodiment two or more peptides or peptide variants of theinvention are encoded and thus expressed in a successive order (similarto “beads on a string” constructs). In doing so, the peptides or peptidevariants may be linked or fused together by stretches of linker aminoacids, such as for example LLLLLL, or may be linked without anyadditional peptide(s) between them. These constructs can also be usedfor cancer therapy and may induce immune responses both involving MHC Iand MHC II.

The present invention also relates to a host cell transformed with apolynucleotide vector construct of the present invention. The host cellcan be either prokaryotic or eukaryotic. Bacterial cells may bepreferred prokaryotic host cells in some circumstances and typically area strain of E. coli such as, for example, the E. coli strains DH5available from Bethesda Research Laboratories Inc., Bethesda, MD, USA,and RR1 available from the American Type Culture Collection (ATCC) ofRockville, MD, USA (No ATCC 31343). Preferred eukaryotic host cellsinclude yeast, insect and mammalian cells, preferably vertebrate cellssuch as those from a mouse, rat, monkey or human fibroblastic and coloncell lines. Yeast host cells include YPH499, YPH500 and YPH501, whichare generally available from Stratagene Cloning Systems, La Jolla, CA92037, USA. Preferred mammalian host cells include chinese hamster ovary(CHO) cells available from the ATCC as CCL61, NIH Swiss mouse embryocells NIH/3T3 available from the ATCC as CRL 1658, monkey kidney-derivedCOS-1 cells available from the ATCC as CRL 1650 and 293 cells which arehuman embryonic kidney cells. Preferred insect cells are Sf9 cells whichcan be transfected with baculovirus expression vectors. An overviewregarding the choice of suitable host cells for expression can be foundin the literature (Balbás and Lorence, 2004).

Transformation of appropriate cell hosts with a DNA construct of thepresent invention is accomplished by well-known methods that typicallydepend on the type of vector used. With regard to transformation ofprokaryotic host cells, see for example Cohen et al. or Green andSambrook (Cohen et al., 1972; Green and Sambrook, 2012). Transformationof yeast cells is described in Sherman et al. (Sherman et al., 1986).The method of Beggs (Beggs, 1978) is also useful. With regard tovertebrate cells, reagents useful in transfecting such cells, forexample calcium phosphate and DEAE-dextran or liposome formulations, areavailable from Stratagene Cloning Systems, or Life Technologies Inc.,Gaithersburg, MD 20877, USA. Electroporation is also useful fortransforming and/or transfecting cells and is well known in the art fortransforming yeast cell, bacterial cells, insect cells and vertebratecells. The contents of each of these references is herein incorporatedby reference in their entirety.

Successfully transformed cells, i.e. cells that contain a DNA constructof the present invention, can be identified by well-known techniquessuch as PCR. Alternatively, the presence of the protein in thesupernatant can be detected using antibodies.

It will be appreciated that certain host cells of the invention areuseful in the preparation of the peptides of the invention, for examplebacterial, yeast and insect cells. However, other host cells may beuseful in certain therapeutic methods. For example, antigen-presentingcells, such as dendritic cells, may usefully be used to express thepeptides of the invention such that they may be loaded into appropriateMHC molecules. Thus, the current invention provides a host cellcomprising a nucleic acid or an expression vector according to theinvention.

In a preferred embodiment the host cell is an antigen presenting cell,in particular a dendritic cell or antigen presenting cell. APCs loadedwith a recombinant fusion protein containing prostatic acid phosphatase(PAP) were approved by the U.S. Food and Drug Administration (FDA) onApr. 29, 2010, to treat asymptomatic or minimally symptomatic metastaticHRPC (Sipuleucel-T) (Rini et al., 2006; Small et al., 2006, the contentsof which are incorporated by reference in their entirety).

A further aspect of the invention provides a method of producing apeptide or its variant, the method comprising culturing a host cell andisolating the peptide from the host cell or its culture medium.

In another embodiment, the peptide, the nucleic acid or the expressionvector of the invention are used in medicine. For example, the peptideor its variant may be prepared for i.v., s.c., i.d., i.p., i.m.injection. Preferred methods of peptide injection include s.c., i.d.,i.p., i.m., and i.v. Preferred methods of DNA injection include i.d.,i.m., s.c., i.p. and i.v. Doses of e.g. between 50 μg and 1.5 mg,preferably 125 μg to 500 μg, of peptide or DNA may be given and willdepend on the respective peptide or DNA. Dosages of this range weresuccessfully used in previous trials (Walter et al., 2012).

The polynucleotide used for active vaccination may be substantially pureor contained in a suitable vector or delivery system. The nucleic acidmay be DNA, cDNA, PNA, RNA or a combination thereof. Methods fordesigning and introducing such a nucleic acid are well known in the art.An overview is provided by Teufel et al. (Teufel et al., 2005, thecontents of which are incorporated by reference in their entirety).Polynucleotide vaccines are easy to prepare, but the mode of action ofthese vectors in inducing an immune response is not fully understood.Suitable vectors and delivery systems include viral DNA and/or RNA, suchas systems based on adenovirus, vaccinia virus, retroviruses, herpesvirus, adeno-associated virus or hybrids containing elements of morethan one virus. Non-viral delivery systems include cationic lipids andcationic polymers and are well known in the art of DNA delivery.Physical delivery, such as via a “gene-gun” may also be used. Thepeptide or peptides encoded by the nucleic acid may be a fusion protein,for example with an epitope that stimulates T cells for the respectiveopposite CDR as noted above.

The medicament of the invention may also include one or more adjuvants.Adjuvants are substances that non-specifically enhance or potentiate theimmune response e.g. immune responses mediated by CD8-positive T cellsand helper T (TH) cells to an antigen and would thus be considereduseful in the medicament of the present invention.

Suitable adjuvants include, but are not limited to, 1018 ISS, aluminumsalts, AMPLIVAX®, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellinor TLR5 ligands derived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31,Imiquimod (ALDARA®), Resiquimod, ImuFact IMP321, Interleukins as IL-2,IL-13, IL-21, Interferon-alpha or -beta, or pegylated derivativesthereof, IS Patch, ISS, ISCOMATRIX, ISCOMs, Juvlmmune®, LipoVac, MALP2,MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206,Montanide ISA 50V, Montanide ISA-51, water-in-oil and oil-in-wateremulsions, OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vectorsystem, poly(lactid co-glycolid) [PLG]-based and dextran microparticles,talactoferrin SRL172, Virosomes and other Virus-like particles, YF-17D,VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, which isderived from saponin, mycobacterial extracts and synthetic bacterialcell wall mimics, and other proprietary adjuvants such as Ribi's Detox,Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred.Several immunological adjuvants (e.g. MF59) specific for dendritic cellsand their preparation have been described previously (Allison andKrummel, 1995, the contents of which are incorporated by reference intheir entirety).

Cytokines may also be used. Several cytokines have been directly linkedto influencing dendritic cell migration to lymphoid tissues (e.g., TNF),accelerating the maturation of dendritic cells into efficientantigen-presenting cells for T-lymphocytes (e.g., GM-CSF, IL-1 and IL-4)(U.S. Pat. No. 5,849,589, incorporated herein by reference in itsentirety) and acting as immunoadjuvants (e.g., IL-2, IL-7, IL-12, IL-15,IL-21, IL-23, IFN-alpha. IFN-beta) (Gabrilovich et al., 1996, thecontents of which are incorporated by reference in their entirety). Inan aspect, cytokines and immunological adjuvants may be used in vitro,such as for expansion or activation of T cells, or for ex vivo uses.

CpG immunostimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine setting. Without beingbound by theory, CpG oligonucleotides act by activating the innate(non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9.CpG triggered TLR9 activation enhances antigen specific humoral andcellular responses to a wide variety of antigens, including peptide orprotein antigens, live or killed viruses, dendritic cell vaccines,autologous cellular vaccines and polysaccharide conjugates in bothprophylactic and therapeutic vaccines. More importantly it enhancesdendritic cell maturation and differentiation, resulting in enhancedactivation of TH1 cells and strong cytotoxic T-lymphocyte (CTL)generation, even in the absence of CD4 T cell help. The TH1 bias inducedby TLR9 stimulation is maintained even in the presence of vaccineadjuvants such as alum or incomplete Freund's adjuvant (IFA) thatnormally promote a TH2 bias. CpG oligonucleotides show even greateradjuvant activity when formulated or co-administered with otheradjuvants or in formulations such as microparticles, nanoparticles,lipid emulsions or similar formulations, which are especially necessaryfor inducing a strong response when the antigen is relatively weak. Theyalso accelerate the immune response and enable the antigen doses to bereduced by approximately two orders of magnitude, with comparableantibody responses to the full-dose vaccine without CpG in someexperiments (Krieg, 2006). U.S. Pat. No. 6,406,705 B1, which isincorporated by reference in its entirety, describes the combined use ofCpG oligonucleotides, non-nucleic acid adjuvants and an antigen toinduce an antigen specific immune response. A CpG TLR9 antagonist isdSLIM (double Stem Loop Immunomodulator) by Mologen (Berlin, Germany)which is a preferred component of the pharmaceutical composition of thepresent invention. Other TLR binding molecules such as RNA binding TLR7, TLR 8 and/or TLR 9 may also be used.

Other examples for useful adjuvants include, but are not limited to,chemically modified CpGs (e.g. CpR, Idera), dsRNA analogues such asPoly(I:C) and derivates thereof (e.g. AmpliGen®, Hiltonol®, poly(ICLC),poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA as well asimmunoactive small molecules and antibodies such as cyclophosphamide,sunitinib, immune checkpoint inhibitors including ipilimumab, nivolumab,pembrolizumab, atezolizumab, avelumab, durvalumab, and cemiplimab,Bevacizumab®, Celebrex, NCX-4016, sildenafil, tadalafil, vardenafil,sorafenib, temozolomide, temsirolimus, XL-999, CP-547632, pazopanib,VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other antibodies targeting keystructures of the immune system (e.g. anti-CD40, anti-TGF-beta,anti-TNF-alpha receptor) and SC58175, which may act therapeuticallyand/or as an adjuvant. The amounts and concentrations of adjuvants andadditives useful in the context of the present invention can readily bedetermined by the skilled artisan without undue experimentation.

Preferred adjuvants are anti-CD40, imiquimod, Resiquimod, GM-CSF,cyclophosphamide, sunitinib, bevacizumab, interferon-alpha, CpGoligonucleotides and derivates, Poly(I:C) and derivates, RNA,sildenafil, and particulate formulations with PLG or virosomes.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as GM-CSF, sargramostim,cyclophosphamide, imiquimod, Resiquimod, interferon-alpha, or mixturesthereof.

In a preferred embodiment of the pharmaceutical composition according tothe invention, the adjuvant is cyclophosphamide, imiquimod orResiquimod. Even more preferred adjuvants are Montanide IMS 1312,Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, poly-ICLC(Hiltonol®) and anti-CD40 mAb, or combinations thereof.

This composition is used for parenteral administration, such assubcutaneous, intradermal, intramuscular or oral administration. Thecomposition may be administered via subcutaneous, intramuscular,intravenous, intraperitoneal, intrapleural, intravesicular, intrathecal,topical, oral administration, or a combination of routes. For this, thepeptides and optionally other molecules are dissolved or suspended in apharmaceutically acceptable, preferably aqueous carrier. In addition,the composition can contain excipients, such as buffers, binding agents,blasting agents, diluents, flavors, lubricants, etc. Pharmaceuticallyacceptable carriers include, but are not limited to, excipient,lubricant, emulsifier, stabilizer, solvent, diluent, buffer, vehicle, ora combination thereof. The peptides or T cells recognizing the peptideof the present disclosure in a complex with an MHC molecule can also beadministered together with immune stimulating substances, such ascytokines shown in table 6.

TABLE 6 Immune stimulating cytokines Cytokines EOTAXIN IL-15 G-CSF IL-17GM-CSF IP-10 INF-γ MIP-2 IL-1α KC M-CSF LIF IL-1β LIX IL-2 MCP-1 IL-3MIP-1α IL-4 MIP-1β IL-5 MIG IL-6 RANTES IL-7 TNFα IL-10 IL-12 (P70)IL-12 (p40) VEGF IL-123 IL-9 IL-18 IL-21

Cytokines, e.g., IL-2, IL-7, IL-12, IL-15, IL-21, IFN-α, and IFN-β, mayalso be used in the activation and/or expansion of T cells, such as Tcells recognizing the peptide of the present disclosure in a complexwith an MHC molecule.

An extensive listing of excipients that can be used in such acomposition, can be, for example, taken from A. Kibbe, Handbook ofPharmaceutical Excipients (Kibbe, 2000). Other examples of suitablepharmaceutical carriers are described in Remington's PharmaceuticalSciences (Gennaro, 1997; Banker and Rhodes, 2002, the contents of whichare herein incorporated by reference in their entireties). Thecomposition can be used for a prevention, prophylaxis and/or therapy ofadenomatous or cancerous diseases. Exemplary formulations can be forexample found in EP2112253.

It is important to realize that the immune response triggered by thevaccine according to the invention attacks the cancer in differentcell-stages and different stages of development. Furthermore, differentcancer associated signaling pathways are attacked. This is an advantageover vaccines that address only one or few targets, which may cause thetumor to easily adapt to the attack (tumor escape). Furthermore, not allindividual tumors express the same pattern of antigens. Therefore, acombination of several tumor-associated peptides ensures that everysingle tumor bears at least some of the targets. The composition isdesigned in such a way that each tumor is expected to express several ofthe antigens and cover several independent pathways necessary for tumorgrowth and maintenance. Thus, the vaccine can easily be used“off-the-shelf” for a larger patient population. This means that apre-selection of patients to be treated with the vaccine can berestricted to HLA typing, does not require any additional biomarkerassessments for antigen expression, but it is still ensured that severaltargets are simultaneously attacked by the induced immune response,which is important for efficacy (Banchereau et al., 2001; Walter et al.,2012, the contents of which are incorporated by reference in theirentirety).

As used herein, the term “scaffold” refers to a molecule thatspecifically binds to an (e.g. antigenic) determinant. In oneembodiment, a scaffold is able to direct the entity to which it isattached (e.g. a (second) antigen binding moiety) to a target site, forexample to a specific type of tumor cell or tumor stroma bearing theantigenic determinant (e.g. the complex of a peptide with MHC, accordingto the application at hand). In another embodiment a scaffold is able toactivate signaling through its target antigen, for example a T cellreceptor complex antigen. Scaffolds include, but are not limited to,antibodies and fragments thereof, antigen binding domains of anantibody, comprising an antibody heavy chain variable region and anantibody light chain variable region, binding proteins comprising atleast one ankyrin repeat motif and single domain antigen binding (SDAB)molecules, aptamers, (soluble) TCRs and (modified) cells such asallogenic or autologous T cells. To assess whether a molecule is ascaffold binding to a target, binding assays can be performed.

“Specific” binding means that the scaffold binds the peptide-MHC complexof interest better than other naturally occurring peptide-MHC complexes,to an extent that a scaffold armed with an active molecule that is ableto kill a cell bearing the specific target is not able to kill anothercell without the specific target but presenting other peptide-MHCcomplex(es). Binding to other peptide-MHC complexes is irrelevant if thepeptide of the cross-reactive peptide-MHC is not naturally occurring,i.e., not derived from the human HLA peptidome. Tests to assess targetcell killing are well known in the art. They should be performed usingtarget cells (primary cells or cell lines) with unaltered peptide-MHCpresentation, or cells loaded with peptides such that naturallyoccurring peptide-MHC levels are reached.

Each scaffold can comprise a labelling which provides that the boundscaffold can be detected by determining the presence or absence of asignal provided by the label. For example, the scaffold can be labelledwith a fluorescent dye or any other applicable cellular marker molecule.Such marker molecules are well known in the art. For example, afluorescence-labelling, for example provided by a fluorescence dye, canprovide a visualization of the bound aptamer by fluorescence or laserscanning microscopy or flow cytometry.

Each scaffold can be conjugated with a second active molecule such asfor example IL-21, anti-CD3, and anti-CD28.

For further information on polypeptide scaffolds see for example thebackground section of WO 2014/071978A1 and the references cited therein,the contents of which are incorporated by reference in their entirety.

The peptides of the present invention can be used to generate anddevelop specific antibodies against MHC-peptide complexes. These can beused for therapy, targeting toxins or radioactive substances to thediseased tissue. Another use of these antibodies can be targetingradionuclides to the diseased tissue for imaging purposes such as PET.This use can help to detect small metastases or to determine the sizeand precise localization of diseased tissues.

Therefore, it is a further aspect of the invention to provide a methodfor producing a recombinant antibody specifically binding to a MHC classI or II molecule being complexed with a HLA restricted antigen(preferably a peptide according to the present invention), the methodcomprising: immunizing genetically engineered non-human mammalcomprising cells expressing said human MHC class I or II molecule with asoluble form of a MHC class I or II molecule being complexed with saidHLA restricted antigen; isolating mRNA molecules from antibody producingcells of said non-human mammal; producing a phage display librarydisplaying protein molecules encoded by said mRNA molecules; andisolating at least one phage from said phage display library, said atleast one phage displaying said antibody specifically binding to saidMHC class I or II molecule being complexed with said HLA restrictedantigen.

It is thus a further aspect of the invention to provide an antibody thatspecifically binds to a MHC class I or II molecule being complexed withan HLA restricted antigen, wherein the antibody preferably is apolyclonal antibody, monoclonal antibody, bispecific antibody, achimeric antibody, antibody fragments thereof, or a combination thereof.

Bispecific Antibody

In an aspect, a bispecific antibody includes an antibody capable ofselectively binding two or more epitopes. Bispecific antibodies may bemanufactured in a variety of ways (Holliger & Winter, 1993, the contentsof which are incorporated by reference in their entirety), for instance,prepared chemically or from hybrid hybridomas, or may be any of thebispecific antibody fragments mentioned above. scFv dimers or diabodiesmay be used, rather than whole antibodies. Diabodies and scFv can beconstructed without an Fc region, using only variable domains (usuallyincluding the variable domain components from both light and heavychains of the source antibody), potentially reducing the effects ofanti-idiotypic reaction. Other forms of bispecific antibodies includethe single chain “Janusins” described by Traunecker and colleagues(Traunecker et al., 1991, the contents of which are incorporated byreference in their entirety).

Bispecific antibodies generally include two different binding domains,with each binding domain specifically binding a different epitope eitheron two different antigens or on the same antigen. If a bispecificantibody is capable of selectively binding two different epitopes (afirst epitope and a second epitope), the affinity of the first bindingfor the first epitope will generally be at least one to two or three orfour orders of magnitude lower than the affinity of the first bindingdomain for the second epitope, and vice versa. The epitopes recognizedby the bispecific antibody can be on the same or a different target(e.g., on the same or a different protein). Bispecific antibodies can bemade for example by combining binding domains that recognize differentepitopes of the same antigen.

Some example bispecific antibodies have two heavy chains (each havingthree heavy chain CDRs, followed by (N-terminal to C-terminal) a CH1domain, a hinge, a CH2 domain, and a CH3 domain), and two immunoglobulinlight chains that confer antigen-binding specificity through associationwith each heavy chain. However, additional architectures are envisioned,including bispecific antibodies in which the light chain(s) associatewith each heavy chain but do not (or minimally) contribute toantigen-binding specificity, or that can bind one or more of theepitopes bound by the heavy chain antigen-binding regions, or that canassociate with each heavy chain and enable binding of one or both of theheavy chains to one or both epitopes.

In particular embodiments, a bispecific antibody can include an antibodyarm combined with an arm that binds to a triggering molecule on aleukocyte, such as a T cell receptor molecule (for example, CD3), or Fcreceptors for IgG (Fc gamma R), such as Fc gamma RI (CD64), Fc gamma RII(CD32) and Fc gamma RIII (CD 16), so as to focus and localize cellulardefense mechanisms to the targeted disease cell. Bispecific antibodiesalso can be used to localize cytotoxic agents to targeted disease cells.

Bispecific antibodies can be prepared as full-length antibodies orantibody fragments (for example, F(ab′)₂ bispecific antibodies). Seee.g. WO 1996/016673; U.S. Pat. No. 5,837,234; WO 1998/002463; U.S. Pat.No. 5,821,337, the contents of which are incorporated by reference intheir entirety.

A bispecific antibody can have an extended half-life. In particularembodiments, half-life extension of a bispecific antibody can beachieved by: increasing the hydrodynamic volume of the antibody bycoupling to inert polymers such as polyethylene glycol or other mimetichydrophilic polymers; fusion or conjugation to large disorderedpeptides; or fusing or coupling the antibody to a ligand. Thesealterations and a number of others are described elsewhere (U.S. Pat.Nos. 7,083,784, 7,670,600, U.S. Patent Application Publication No.2010/0234575, and Zwolak et al., 2017, the contents of which areincorporated by reference in their entirety). Bispecific antibodies withextended half-lives are described in for example U.S. Pat. No. 8,921,528and U.S. Patent Application Publication No. 2014/0308285, the contentsof which are incorporated by reference in their entirety.

Methods for making bispecific antibodies are known in the art.Production of full-length bispecific antibodies is based on theco-expression of two immunoglobulin heavy chain-light chain pairs, wherethe two chains have different specificities. See e.g., WO 1993/008829and Traunecker et al., 1991, the contents of which are incorporated byreference in their entirety.

Polyclonal Antibody

Methods for making polyclonal antibodies are known in the art.Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen.Polyclonal antibodies which selectively bind a peptide according to SEQID NO: 1 to SEQ ID NO: 102, or a variant or fragment thereof may be madeby methods well-known in the art (see, e.g., Howard & Kaser, 2007, thecontents of which are incorporated by reference in their entirety.

Chimeric Antibody

Chimeric antibodies are molecules different portions of which arederived from different animal species, such as those having variableregion derived from a murine antibody and a human immunoglobulinconstant region, which are primarily used to reduce immunogenicity inapplication and to increase yields in production for example wheremurine monoclonal antibodies have higher yields from hybridomas buthigher immunogenicity in humans, such that human murine chimericmonoclonal antibodies are used. Chimeric antibodies and methods fortheir production are known in the art (Cabilly et al., 1984; Morrison etal., 1984; Boulianne et al., 1984; European Patent Application 173494(1986); WO 86/01533 (1986); European Patent Application 184187 (1986);Sahagan et al., 1986; Liu et al., 1987; Sun et al., 1987; Better et al.,1988; Harlow & Lane, 1998; U.S. Pat. No. 5,624,659, the contents ofwhich are incorporated by reference in their entirety).

Antibody Fragments

In addition to entire immunoglobulins (or their recombinantcounterparts), immunoglobulin fragments comprising the epitope bindingsite (e.g., Fab′, F(ab′)₂, or other fragments) may be synthesized.“Fragments” or minimal immunoglobulins may be designed utilizingrecombinant immunoglobulin techniques. For instance, “Fv”immunoglobulins for use in the present invention may be produced bysynthesizing a fused variable light chain region and a variable heavychain region. Combinations of antibodies are also of interest, e.g.,diabodies, which comprise two distinct Fv specificities. Antigen-bindingfragments of immunoglobulins include, but are not limited to, SMIPs(small molecule immunopharmaceuticals), camelbodies, nanobodies, andIgNAR.

Respective methods for producing such antibodies and single chain MHCclass I complexes, as well as other tools for the production of theseantibodies are disclosed in WO 03/068201, WO 2004/084798, WO 01/72768,WO 03/070752, and in publications (Cohen et al., 2003a; Cohen et al.,2003b; Denkberg et al., 2003, which for the purposes of the presentinvention are incorporated by reference in their entireties).

Preferably, the antibody is binding with a binding affinity of <100 nM,more preferably <50 nM, more preferably <10 nM, more preferably <1 nM,more preferably <0.1 nM, more preferably <0.01 nM, to the complex, whichis also regarded as “specific” in the context of the present invention.

The present invention relates to a peptide comprising an amino acidsequence selected from the group consisting of

-   -   SEQ ID NO: 1 to SEQ ID NO: 102,    -   and a variant sequence thereof which maintains capacity to bind        to MHC molecule(s) and/or induce T cells cross-reacting with        said variant peptide,

or a pharmaceutically acceptable salt thereof.

The peptides disclosed bind to HLA-A*02 allotype MHC molecules.Likewise, the peptide variant's capacity to bind to MHC molecule(s)relates to HLA-A*02 allotype MHC molecules.

In one embodiment, variant sequences of the claimed peptides in theabove meaning are sequences which have substitutions in their so-called“anchoring position”.

Note that these anchoring positions in MHC restricted peptides compriseamino acid residues which mediate binding of the peptide to the peptidebinding groove in the MHC. They play only a minor role in the bindingreaction between the binding polypeptide and the peptide-MHC complex,meaning that substitutions in these positions do not significantlyaffect immunogenicity or TCR/antibody binding of a peptide modified insuch way.

For HLA-A*02, the anchoring positions are in position 2 (P2) andposition 9 (P9) of the respective MHC restricted peptide (see table 7).In P2, the preferred amino acid residues are most often leucin (L) ormethionine (M), while in P9, the preferred amino acid residues areleucin (L) or valine (V).

TABLE 7 Anchoring position and preferred amino acids in these positionsfor HLA-A*02. Accepted Accepted HLA Anchor amino acid Anchor amino acidsubtype position 1 residues position 2 residues HLA-A*02 Position 2 L orM Position 9 L or V

Furthermore, the invention relates to variants thereof which are atleast 88% homologous to SEQ ID NO: 1 to SEQ ID NO: 102, provided theybinds to MHC molecule(s) and/or induce T cells cross-reacting with saidvariant peptide.

In one embodiment, variant sequences of the claimed peptides in theabove meaning are sequences which are modified by at least oneconservative amino acid substitution. The definition and scope of theterm “conservative amino acid substitution” is disclosed in table 3 and4, and the description related thereto.

In one embodiment, said peptide has the ability to bind to an MHC classI molecule, and, when bound to said MHC, is capable of being recognizedby CD4 and/or CD8 T cells.

In one embodiment, the MHC class I molecule is an HLA-A*02 allotype MHCmolecule.

The present invention further relates to a peptide comprising a sequencethat is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:102 or a variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 102, wherein said peptide orvariant has an overall length of between 8 and 30, and preferred between8 and 12 amino acids or between 9 and 30, and preferred between 9 and 12amino acids if the selected peptide has a length of 9 amino acids orbetween 10 and 30, and preferred between 10 and 12 amino acids if theselected peptide has a length of 10 amino acids.

In one embodiment said peptide or variant thereof comprises 1 to 4additional amino acids at the C- and/or N-terminus of the respectivesequence. See table 5 for further details. In one embodiment, saidpeptide or variant thereof has a length of up to 30 amino acids. In oneembodiment, said peptide or variant thereof has a length of up to 16amino acids. In one embodiment, said peptide or variant thereof has alength of up to 12 amino acids.

In one embodiment, said peptide or variant thereof has an overall lengthfrom 8 to 30 amino acids. In one embodiment, said peptide or variantthereof has an overall length from 8 to 16 amino acids. In oneembodiment, said peptide or variant thereof has an overall length from 8to 12 amino acids.

In one embodiment, said peptide or variant thereof has an overall lengthfrom 9 to 30 amino acids. In one embodiment, said peptide or variantthereof has an overall length from 9 to 16 amino acids. In oneembodiment, said peptide or variant thereof has an overall length from 9to 12 amino acids.

In one embodiment, said peptide or variant thereof has an overall lengthfrom 10 to 30 amino acids. In one embodiment, said peptide or variantthereof has an overall length from 10 to 16 amino acids. In oneembodiment, said peptide or variant thereof has an overall length from10 to 12 amino acids.

In one embodiment, said peptide or variant thereof has a lengthaccording to the respective SEQ ID NO: 1 to SEQ ID NO: 102. In oneembodiment, the peptide consists or consists essentially of the aminoacid sequence according to SEQ ID NO: 1 to SEQ ID NO: 102.

The present invention further relates to the peptides according to theinvention, wherein the peptide includes non-peptide bonds.

The present invention further relates to the peptides according to theinvention, wherein the peptide is part of a fusion protein, inparticular comprising N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii), or wherein the peptide is fusedto (or into) an antibody, such as for example an antibody that isspecific for dendritic cells. The present invention further relates toan antibody, or a functional fragment thereof, that specificallyrecognizes or binds to the peptide or variant thereof according to thepresent invention, or to the peptide or variant thereof according to thepresent invention when bound to an MHC molecule.

In one embodiment, the MHC molecule is an HLA-A*02 allotype MHCmolecule.

In further embodiments, such antibody is soluble or membrane bound. Infurther embodiments, such antibody is a monoclonal antibody or fragmentthereof. In further embodiments, such antibody carries a furthereffector function such as an immune stimulating domain or toxin.

The present invention further relates to a T cell receptor, or afunctional fragment thereof, that is reactive with, or binds to, an MHCligand, wherein said ligand is the peptide or variant thereof accordingto the present invention, or the peptide or variant thereof according tothe present invention when bound to an MHC molecule.

In one embodiment, the MHC molecule is an HLA-A*02 allotype MHCmolecule. In further embodiments, said T cell receptor is provided as asoluble molecule. In further embodiments, said T cell receptor carries afurther effector function such as an immune stimulating domain or toxin.

The present invention further relates to a nucleic acid, encoding for apeptide or variant thereof according to the invention, an antibody orfragment thereof according to the invention, or a T cell receptor orfragment thereof according to the invention.

The present invention further relates to the nucleic acid according tothe invention that is DNA, cDNA, PNA, RNA or combinations thereof.

In one embodiment, said nucleic acid is linked to a heterologouspromoter sequence. In one embodiment, said nucleic acid is provided asan expression vector expressing and/or comprising said nucleic acid.

The present invention further relates to a recombinant host cellcomprising a peptide or variant thereof according to the invention, anantibody or fragment thereof according to the invention, a T cellreceptor or fragment thereof according to the invention, or a nucleicacid or expression vector according to the invention.

The present invention further relates to an in vitro method forproducing activated T lymphocytes, the method comprising contacting invitro T cells with antigen loaded human class I or II MHC moleculesexpressed on the surface of a suitable antigen-presenting cell or anartificial construct mimicking an antigen-presenting cell for a periodof time sufficient to activate said T cells in an antigen specificmanner, wherein said antigen is a peptide or variant thereof accordingto the respective description.

In one embodiment, the antigen-presenting cell comprises an expressionvector capable of expressing said peptide containing SEQ ID NO: 1 to SEQID NO: 102 or said variant amino acid sequence.

The present invention further relates to an in vitro method of producinga peptide according to the present invention, said method comprisingculturing the host cell according to the present invention, andisolating the peptide from said host cell or its culture medium.

The present invention further relates to an activated T lymphocyte,produced by the method according to the present invention, wherein saidT lymphocyte selectively recognizes a cell which presents a peptide orvariant thereof according to the present invention. Said presentationcan be an aberrant presentation or aberrant expression.

The present invention further relates to pharmaceutical compositioncomprising at least one active ingredient selected from the groupconsisting of

-   -   the peptide or variant thereof according to the present        invention,    -   the antibody or fragment thereof according to the present        invention,    -   the T cell receptor or fragment thereof according to the present        invention,    -   the nucleic acid or the expression vector according to the        present invention,    -   the host cell according to the present invention,    -   or the activated T lymphocyte according to the present        invention,

and a pharmaceutically acceptable carrier.

In one embodiment, such pharmaceutical composition is personalizedpharmaceutical composition for an individual patient. In one embodiment,the pharmaceutical composition is a vaccine. The method could also beadapted to produce T cell clones for down-stream applications, such asTCR isolations, or soluble antibodies, and other treatment options.

A “personalized pharmaceutical” shall mean specifically tailoredtherapies for one individual patient that will only be used for therapyin such individual patient, including actively personalized cancervaccines and adoptive cellular therapies using autologous patienttissue.

The present invention further relates to a method for producing thepeptide or variant thereof according to the present invention, theantibody or fragment thereof according to the present invention, or theT cell receptor or fragment thereof according to the present invention,and isolating the peptide or variant thereof, the antibody or fragmentthereof or the T cell receptor or fragment thereof from said host celland/or its culture medium.

The present invention further relates to a peptide or variant thereofaccording to the present invention, the antibody or fragment thereofaccording to the present invention, the T cell receptor or fragmentthereof according to the present invention, the nucleic acid or theexpression vector according to the present invention, the host cellaccording to the present invention, or the activated T lymphocyteaccording to the present invention, for use in medicine, or for use inthe manufacture of a medicine.

The present invention further relates to a method of killing targetcells in a patient, which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention.Said method comprises administering to the patient an effective numberof activated T lymphocytes as according to the present invention.

Likewise, the present invention relates to an activated T lymphocyteaccording to the present invention for use in the killing of targetcells in a patient, which target cells present a polypeptide comprisingany amino acid sequence according to the present invention, or for usein the manufacture of a medicament for the killing of such target cells.

The present invention further relates to method of treating a patient

-   -   being diagnosed for,    -   suffering from or    -   being at risk of developing

cancer, the method comprising administering to the patient an effectiveamount of the peptide or variant thereof according to the presentinvention, the antibody or fragment thereof according to the presentinvention, the T cell receptor or fragment thereof according to thepresent invention, the nucleic acid or the expression vector accordingto the present invention, the host cell according to the presentinvention, or the activated T lymphocyte according to the presentinvention.

Likewise, the present invention further relates to the peptide orvariant thereof according to the present invention, the antibody orfragment thereof according to the present invention, the T cell receptoror fragment thereof according to the present invention, the nucleic acidor the expression vector according to the present invention, the hostcell according to the present invention, or the activated T lymphocyteaccording to the present invention, for use the treatment of a patient

-   -   being diagnosed for,    -   suffering from or    -   being at risk of developing

cancer, or for use in the manufacture of a medicament for the treatmentof such patient.

The present invention further relates to a use according to theinvention, wherein the medicament is a vaccine.

The present invention further relates to the method or the peptide,antibody, T cell receptor, nucleic acid, host cell or activated Tlymphocyte for use according to the invention, wherein said cancer isselected from the group consisting of acute myeloid leukemia, breastcancer, cholangiocellular carcinoma, chronic lymphocytic leukemia,colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer,gastro-esophageal junction cancer, hepatocellular carcinoma, head andneck squamous cell carcinoma, melanoma, non-Hodgkin lymphoma, non-smallcell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, small cell lung cancer, urinarybladder carcinoma, and uterine endometrial cancer.

The present invention further relates to a kit comprising:

-   -   (a) a container comprising a pharmaceutical composition        containing the pharmaceutical composition according to the        present invention in solution or in lyophilized form;    -   (b) optionally, a second container containing a diluent or        reconstituting solution for the lyophilized formulation;    -   (c) optionally, at least one more peptide selected from the        group consisting of SEQ ID NO: 1 to SEQ ID NO: 102.

In further embodiments, the kit comprises one or more of a buffer, adiluent, a filter, a needle, or a syringe.

The present invention further relates to particular marker proteins andbiomarkers based on the peptides according to the present invention,herein called “targets” that can be used in the diagnosis and/orprognosis of at least one of acute myeloid leukemia, breast cancer,cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectalcancer, gallbladder cancer, glioblastoma, gastric cancer,gastro-esophageal junction cancer, hepatocellular carcinoma, head andneck squamous cell carcinoma, melanoma, non-Hodgkin lymphoma, non-smallcell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, small cell lung cancer, urinarybladder carcinoma, and uterine endometrial cancer.

The term “antibody” or “antibodies” is used herein in a broad sense andincludes both polyclonal and monoclonal antibodies. In addition tointact or “full” immunoglobulin molecules, also included in the term“antibodies” are fragments (e.g. CDRs, Fv, Fab and Fc fragments) orpolymers of those immunoglobulin molecules and humanized versions ofimmunoglobulin molecules, as long as they exhibit any of the desiredproperties (e.g., specific binding of acute myeloid leukemia, breastcancer, cholangiocellular carcinoma, chronic lymphocytic leukemia,colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer,gastro-esophageal junction cancer, hepatocellular carcinoma, head andneck squamous cell carcinoma, melanoma, non-Hodgkin lymphoma, non-smallcell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, small cell lung cancer, urinarybladder carcinoma, and uterine endometrial cancer marker polypeptide)according to the invention.

Whenever possible, the antibodies of the invention may be purchased fromcommercial sources. The antibodies of the invention may also begenerated using well-known methods. The skilled artisan will understandthat either full length of acute myeloid leukemia, breast cancer,cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectalcancer, gallbladder cancer, glioblastoma, gastric cancer,gastro-esophageal junction cancer, hepatocellular carcinoma, head andneck squamous cell carcinoma, melanoma, non-Hodgkin lymphoma, non-smallcell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, small cell lung cancer, urinarybladder carcinoma, and uterine endometrial cancer marker polypeptides orfragments thereof may be used to generate the antibodies of theinvention. A polypeptide to be used for generating an antibody of theinvention may be partially or fully purified from a natural source ormay be produced using recombinant DNA techniques.

For example, a cDNA encoding a peptide according to the presentinvention, such as a peptide according to SEQ ID NO: 1 to SEQ ID NO: 102polypeptide, or a variant or fragment thereof, can be expressed inprokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g., yeast,insect, or mammalian cells), after which the recombinant protein can bepurified and used to generate a monoclonal or polyclonal antibodypreparation that specifically bind to the acute myeloid leukemia, breastcancer, cholangiocellular carcinoma, chronic lymphocytic leukemia,colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer,gastro-esophageal junction cancer, hepatocellular carcinoma, head andneck squamous cell carcinoma, melanoma, non-Hodgkin lymphoma, non-smallcell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, small cell lung cancer, urinarybladder carcinoma, and uterine endometrial cancer marker polypeptideused to generate the antibody according to the invention.

One of skill in the art will realize that the generation of two or moredifferent sets of monoclonal or polyclonal antibodies maximizes thelikelihood of obtaining an antibody with the specificity and affinityrequired for its intended use (e.g., ELISA, immunohistochemistry, invivo imaging, immunotoxin therapy). The antibodies are tested for theirdesired activity by known methods, in accordance with the purpose forwhich the antibodies are to be used (e.g., ELISA, immunohistochemistry,immunotherapy, etc. (Greenfield, 2014, the contents of which areincorporated by reference in their entirety)). For example, theantibodies may be tested in ELISA assays or, Western blots,immunohistochemical staining of formalin-fixed cancers or frozen tissuesections. After their initial in vitro characterization, antibodiesintended for therapeutic or in vivo diagnostic use are tested accordingto known clinical testing methods.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The monoclonal antibodies herein specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired antagonistic activity (U.S. Pat. No. 4,816,567, which is herebyincorporated by reference in its entirety).

Monoclonal antibodies of the invention may be prepared using hybridomamethods. In a hybridoma method, a mouse or other appropriate host animalis typically immunized with an immunizing agent to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies).

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 and U.S. Pat. No.4,342,566, the contents of which are incorporated by reference in theirentirety. Papain digestion of antibodies typically produces twoidentical antigen binding fragments, called Fab fragments, each with asingle antigen binding site, and a residual Fc fragment. Pepsintreatment yields a F(ab′)2 fragment and a pFc′ fragment.

The antibody fragments, whether attached to other sequences or not, canalso include insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the fragment is not significantly altered orimpaired compared to the non-modified antibody or antibody fragment.These modifications can provide for some additional property, such as toremove/add amino acids capable of disulfide bonding, to increase itsbio-longevity, to alter its secretory characteristics, etc. In any case,the antibody fragment must possess a bioactive property, such as bindingactivity, regulation of binding at the binding domain, etc. Functionalor active regions of the antibody may be identified by mutagenesis of aspecific region of the protein, followed by expression and testing ofthe expressed polypeptide. Such methods are readily apparent to askilled practitioner in the art and can include site-specificmutagenesis of the nucleic acid encoding the antibody fragment.

The antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′ or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed by substituting rodent CDRs or CDR sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567,the contents of which are incorporated by reference in their entirety),wherein substantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome CDR residues and possibly some FR residues are substituted byresidues from analogous sites in rodent antibodies.

Transgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production can be employed. For example, ithas been described that the homozygous deletion of the antibody heavychain joining region gene in chimeric and germ-line mutant mice resultsin complete inhibition of endogenous antibody production. Transfer ofthe human germ-line immunoglobulin gene array in such germ-line mutantmice will result in the production of human antibodies upon antigenchallenge. Human antibodies can also be produced in phage displaylibraries.

In an aspect, the antibody of the present disclosure can also beobtained through phage display, or ribosome display, or yeast display,or bacteria display, or Baculovirus display, or mammal cell display, ormRNA display. These methods are all conventional techniques in the art,the specific operations thereof can be seen in corresponding textbooksor operation manuals (Mondon et al., 2008; the content of which ishereby incorporated by reference in its entirety). Using phage displayas an example, separate antibody genes may be inserted into the DNA ofphages, so that the variable regions on the antibody molecules that canbind the antigens may be coupled to the capsid protein of the phage.After the phage infecting E. coli, single stranded DNA may be replicatedin E. coli, and the phage may be reassembled and secreted into theculture medium, while the E. coli may not be lysed. The phage may beco-incubated with target antigens; and after the bound phages areisolated, amplification and purification may be then conducted so thatgreat amounts of clones can be screened. The phage display technique canbe found in the literature (Liu et al., 2004; the contents of which arehereby incorporated by reference in its entirety).

In another aspect, the present disclosure may include methods forproducing a monoclonal antibody using a phage display method.Specifically, mRNA may be prepared from an animal, e.g., rabbits, rats,mice, guinea pigs, hamsters, goats, horses, chickens, sheep, andcamelids (e.g., llamas), immunized by the method for immunizing ananimal, whereupon cDNA may be prepared using the mRNA as a template, sothat a single-chain antibody (scFv) gene encoding only an antibodyvariable region may be prepared. The gene may be cloned to a phagemidvector. E. coli, into which the phagemid vector is transduced, isinfected with phage, so as to express the scFV antibody on the phagecapsid. Screening of the scFv expressed in this way against an antigenprotein or against a peptide-MHC complex may prepare a monoclonal scFVantibody specific to the antigen protein or the peptide-MHC complex.Herein, preparation of mRNA, preparation of cDNA, subcloning to phagemidor transduction to E. coli, phage infection, and screening of amonoclonal scFV antibody specific to an antigen protein or a peptide-MHCcomplex each may be performed by the known method. For example,subcloning of a scFV gene to a phagemid vector containing two elementsconsisting of a gene fragment encoding a leader sequence (signalsequence) and a phage capsid protein III and a replication origin of M13and using of M13 phage as a phage can express a scFV antibody on the M13phage. Further, a phage obtained by screening may be infected to aspecific bacterium and cultured, so that a monoclonal antibody specificto an antigen protein may also be collected in large quantities from theculture. According to the method for producing a monoclonal antibody ofthe present disclosure, not only an scFV antibody but also an antibodyfragment having no constant region, such as a Fab antibody fragment, maybe prepared.

In another aspect, the present disclosure may include phage displaylibraries, in which the heavy and light chain variable regions of anantibody may be synthesized such that they include nearly all possiblespecificities.

In another aspect, the present disclosure may include generation ofphage display libraries containing phage other than M13. Otherbacteriophages, such as lambda phage, may also be useful in the methodof the present disclosure. Lambda phage display libraries have beengenerated, which display peptides encoded by heterologous DNA on theirsurface (Sternberg et al., 1995; the content of which is herebyincorporated by reference in its entirety). Moreover, the method of thepresent disclosure may be extended to include viruses other thanbacteriophage, such as eukaryotic viruses. Eukaryotic viruses may begenerated that encode genes suitable for delivery to a mammal and thatencode and display an antibody capable of targeting a specific cell typeor tissue into which the gene is to be delivered. For example,retroviral vectors have been generated, which display functionalantibody fragments (Russell et al., 1993; the content of which is herebyincorporated by reference in its entirety).

In another aspect, the present disclosure provides methods for producinga recombinant antibody specifically binding to MHC class I or II beingcomplexed with a HLA restricted antigen (preferably a peptide consistingor consisting essentially of an amino acid sequence according to SEQ IDNO: 1 to SEQ ID NO: 102), the method may include immunizing agenetically engineered non-human mammal comprising cells expressing saidMHC class I or II with a soluble form of a MHC class I or II moleculebeing complexed with said HLA restricted antigen; isolating mRNAmolecules from antibody producing cells of said non-human mammal;producing a phage display library displaying protein molecules encodedby said mRNA molecules; and isolating at least one phage from said phagedisplay library, said at least one phage displaying said antibodyspecifically binding to said MHC class I or II being complexed with saidHLA restricted antigen.

Antibodies of the invention are preferably administered to a subject ina pharmaceutically acceptable carrier. Typically, an appropriate amountof a pharmaceutically acceptable salt is used in the formulation torender the formulation isotonic. Examples of the pharmaceuticallyacceptable carrier include saline, Ringer's solution and dextrosesolution. The pH of the solution is preferably from about 5 to about 8,and more preferably from about 7 to about 7.5. Further carriers includesustained release preparations such as semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, liposomes or microparticles. Itwill be apparent to those persons skilled in the art that certaincarriers may be more preferable depending upon, for instance, the routeof administration and concentration of antibody being administered.

The antibodies can be administered to the subject, patient, or cell byinjection (e.g., intravenous, intraperitoneal, subcutaneous,intramuscular), or by other methods such as infusion that ensure itsdelivery to the bloodstream in an effective form. The antibodies mayalso be administered by intratumoral or peritumoral routes, to exertlocal as well as systemic therapeutic effects. Local or intravenousinjection is preferred.

Effective dosages and schedules for administering the antibodies may bedetermined empirically, and making such determinations is within theskill in the art. Those skilled in the art will understand that thedosage of antibodies that must be administered will vary depending onfor example the subject that will receive the antibody, the route ofadministration, the particular type of antibody used and other drugsbeing administered. A typical daily dosage of the antibody used alonemight range from about 1 μg/kg to up to 100 mg/kg of body weight or moreper day, depending on the factors mentioned above. Followingadministration of an antibody, preferably for treating acute myeloidleukemia, breast cancer, cholangiocellular carcinoma, chroniclymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, gastro-esophageal junction cancer,hepatocellular carcinoma, head and neck squamous cell carcinoma,melanoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovariancancer, esophageal cancer, pancreatic cancer, prostate cancer, renalcell carcinoma, small cell lung cancer, urinary bladder carcinoma, anduterine endometrial cancer, the efficacy of the therapeutic antibody canbe assessed in various ways well known to the skilled practitioner. Forinstance, the size, number, and/or distribution of cancer in a subjectreceiving treatment may be monitored using standard tumor imagingtechniques. A therapeutically-administered antibody that arrests tumorgrowth, results in tumor shrinkage, and/or prevents the development ofnew tumors, compared to the disease course that would occurs in theabsence of antibody administration, is an efficacious antibody fortreatment of cancer.

It is a further aspect of the invention to provide a method forproducing a soluble T cell receptor recognizing a specific peptide-MHCcomplex. Such soluble T cell receptors can be generated from specific Tcell clones, and their affinity can be increased by mutagenesistargeting the complementarity-determining regions. For the purpose of Tcell receptor selection, phage display can be used (US 2010/0113300,Liddy et al., 2012, the contents of which are incorporated by referencein their entirety). For the purpose of stabilization of T cell receptorsduring phage display and in case of practical use as drug, alpha andbeta chains can be linked e.g. by non-native disulfide bonds, othercovalent bonds (single chain T cell receptor), or by dimerizationdomains (Boulter et al., 2003; Card et al., 2004; Willcox et al., 1999,the contents of which are incorporated by reference in their entirety).The T cell receptor can be linked to toxins, drugs, cytokines (see forexample US 2013/0115191, the contents of which are incorporated byreference in their entirety), and domains recruiting effector cells suchas an anti-CD3 domain, etc., in order to execute particular functions ontarget cells. Moreover, it could be expressed in T cells used foradoptive transfer. Further information can be found in WO 2004/033685A1and WO 2004/074322A1. A combination of soluble TCRs is described in WO2012/056407A1. Further methods for the production are disclosed in WO2013/057586A1, the contents of which are incorporated by reference intheir entirety.

In addition, the peptides and/or the TCRs or antibodies or other bindingmolecules of the present invention can be used to verify a pathologist'sdiagnosis of a cancer based on a biopsied sample.

The antibodies or TCRs may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radionucleotide (such as¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P or ³⁵S) so that the tumor can belocalized using immunoscintiography. In one embodiment, antibodies orfragments thereof bind to the extracellular domains of two or moretargets of a protein selected from the group consisting of theabove-mentioned proteins, and the binding affinity (K_(d)) is <100 nM,more preferably <50 nM, more preferably <10 nM, more preferably <1 nM,more preferably <0.1 nM, more preferably <0.01 nM.

Antibodies for diagnostic use may be labeled with probes suitable fordetection by various imaging methods. Methods for detection of probesinclude, but are not limited to, fluorescence, light, confocal andelectron microscopy; magnetic resonance imaging and spectroscopy;fluoroscopy, computed tomography and positron emission tomography.Suitable probes include, but are not limited to, fluorescein, rhodamine,eosin and other fluorophores, radioisotopes, gold, gadolinium and otherlanthanides, paramagnetic iron, fluorine-18 and other positron-emittingradionuclides. Additionally, probes may be bi- or multi-functional andbe detectable by more than one of the methods listed. These antibodiesmay be directly or indirectly labeled with said probes. Attachment ofprobes to the antibodies includes covalent attachment of the probe,incorporation of the probe into the antibody, and the covalentattachment of a chelating compound for binding of probe, amongst otherswell recognized in the art. For immunohistochemistry, the disease tissuesample may be fresh or frozen or may be embedded in paraffin and fixedwith a preservative such as formalin. The fixed or embedded sectioncontains the sample are contacted with a labeled primary antibody andsecondary antibody, wherein the antibody is used to detect theexpression of the proteins in situ.

Another aspect of the present invention includes an in vitro method forproducing activated T cells, the method comprising contacting in vitro Tcells with peptide loaded human MHC molecules expressed on the surfaceof a suitable antigen-presenting cell for a period of time sufficient toactivate the T cell in an antigen specific manner, wherein the antigenis a peptide according to the invention. Preferably, a sufficient amountof the antigen is used with an antigen-presenting cell.

Preferably the mammalian cell lacks or has a reduced level or functionof the TAP peptide transporter. Suitable cells that lack the TAP peptidetransporter include T2, RMA-S and Drosophila cells. TAP is thetransporter associated with antigen processing.

The human peptide loading deficient cell line T2 is available from theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville,Maryland 20852, USA under Catalogue No CRL 1992; the Drosophila cellline Schneider line 2 is available from the ATCC under Catalogue No CRL19863; the mouse RMA-S cell line is described in Ljunggren et al.(Ljunggren and Karre, 1985, the contents of which are incorporated byreference in their entirety).

Preferably, before transfection the host cell expresses substantially noMHC class I molecules. It is also preferred that the stimulator cellexpresses a molecule important for providing a co-stimulatory signal forT cells such as any of B7.1, B7.2, ICAM-1 and LFA3. The nucleic acidsequences of numerous MHC class I molecules and of the co-stimulatormolecules are publicly available from the GenBank and EMBL databases.

In case of an MHC class I epitope being used as an antigen; the T cellsare CD8-positive T cells.

If an antigen-presenting cell is transfected to express such an epitope,preferably the cell comprises an expression vector capable of expressinga peptide containing SEQ ID NO: 1 to SEQ ID NO: 102, or a variant aminoacid sequence thereof.

A number of other methods may be used for generating T cells in vitro.For example, autologous tumor-infiltrating lymphocytes can be used inthe generation of CTL. Plebanski et al. (Plebanski et al., 1995) madeuse of autologous peripheral blood lymphocytes (PLBs) in the preparationof T cells. Furthermore, the production of autologous T cells by pulsingdendritic cells with peptide or polypeptide, or via infection withrecombinant virus is possible. Also, B cells can be used in theproduction of autologous T cells. In addition, macrophages pulsed withpeptide or polypeptide, or infected with recombinant virus, may be usedin the preparation of autologous T cells. S. Walter et al. (Walter etal., 2003, the contents of which are incorporated by reference in theirentirety) describe the in vitro priming of T cells by using artificialantigen presenting cells (aAPCs), which is also a suitable way forgenerating T cells against the peptide of choice. In the presentinvention, aAPCs were generated by the coupling of preformed MHC-peptidecomplexes to the surface of polystyrene particles (microbeads) bybiotin:streptavidin biochemistry. This system permits the exact controlof the MHC density on aAPCs, which allows to selectively elicit high- orlow-avidity antigen specific T cell responses with high efficiency fromblood samples. Apart from MHC-peptide complexes, aAPCs should carryother proteins with co-stimulatory activity like anti-CD28 antibodiescoupled to their surface. Furthermore, such aAPC-based systems oftenrequire the addition of appropriate soluble factors, e. g. cytokines,like interleukin-12.

Allogeneic cells may also be used in the preparation of T cells and amethod is described in detail in WO 97/26328, incorporated herein byreference in its entirety. For example, in addition to Drosophila cellsand T2 cells, other cells may be used to present antigens such as CHOcells, baculovirus-infected insect cells, bacteria, yeast, andvaccinia-infected target cells. In addition, plant viruses may be used,see for example Porta et al. (Porta et al., 1994, the content of whichis incorporated by reference in its entirety)-which describes thedevelopment of cowpea mosaic virus as a high-yielding system for thepresentation of foreign peptides.

The activated T cells that are directed against the peptides of theinvention are useful in therapy. Thus, a further aspect of the inventionprovides activated T cells obtainable by the foregoing methods of theinvention.

Activated T cells, which are produced by the above method, willselectively recognize a cell that aberrantly expresses a polypeptidethat comprises an amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 102.

Preferably, the T cell recognizes the cell by interacting through itsTCR with the HLA-peptide complex (for example, binding). The T cells areuseful in a method of killing target cells in a patient whose targetcells aberrantly express a polypeptide comprising an amino acid sequenceof the invention wherein the patient is administered an effective numberof the activated T cells. The T cells that are administered to thepatient may be derived from the patient and activated as described above(i.e. they are autologous T cells). Alternatively, the T cells are notfrom the patient but are from another individual. Of course, it ispreferred if the individual is a healthy individual. By “healthyindividual” the inventors mean that the individual is generally in goodhealth, preferably has a competent immune system and, more preferably,is not suffering from any disease that can be readily tested for anddetected.

In vivo, the target cells for the CD8-positive T cells according to thepresent invention can be cells of the tumor (which sometimes express MHCclass II) and/or stromal cells surrounding the tumor (which sometimesalso express MHC class II) (Dengjel et al., 2006).

The T cells of the present invention may be used as active ingredientsof a therapeutic composition. Thus, the invention also provides a methodof killing target cells in a patient whose target cells aberrantlyexpress a polypeptide comprising an amino acid sequence of theinvention, the method comprising administering to the patient aneffective number of T cells as defined above.

By “aberrantly expressed” the inventors also mean that the polypeptideis overexpressed compared to levels of expression in normal tissues orthat the gene is silent in the tissue from which the tumor is derivedbut in the tumor it is expressed. By “overexpressed” the inventors meanthat the polypeptide is present at a level at least 1.2-fold of thatpresent in normal tissue; preferably at least 2-fold, and morepreferably at least 5-fold or 10-fold the level present in normaltissue.

T cells may be obtained by methods known in the art, e.g. thosedescribed above. Protocols for this so-called adoptive transfer of Tcells are well known in the art. Several reviews can be found (Gattinoniet al., 2006; Morgan et al., 2006, the contents of which areincorporated by reference in their entirety).

Another aspect of the present invention includes the use of the peptidescomplexed with MHC to generate a T cell receptor whose nucleic acid iscloned and is introduced into a host cell, preferably a T cell. Thisengineered T cell can then be transferred to a patient for therapy ofcancer.

Any molecule of the invention, i.e. the peptide, nucleic acid, antibody,expression vector, cell, activated T cell, T cell receptor or thenucleic acid encoding it, is useful for the treatment of disorders,characterized by cells escaping an immune response. Therefore, anymolecule of the present invention may be used as medicament or in themanufacture of a medicament. The molecule may be used by itself orcombined with other molecule(s) of the invention or (a) knownmolecule(s).

Kits of the present invention preferably comprise a lyophilizedformulation of the present invention in a suitable container andinstructions for its reconstitution and/or use. Suitable containersinclude for example bottles, vials (e.g. dual chamber vials), syringes(such as dual chamber syringes) and test tubes. The container may beformed from a variety of materials such as glass or plastic. Preferablythe kit and/or container contain/s instructions on or associated withthe container that indicates directions for reconstitution and/or use.For example, the label may indicate that the lyophilized formulation isto be reconstituted to peptide concentrations as described above. Thelabel may further indicate that the formulation is useful or intendedfor subcutaneous administration.

The container holding the formulation may be a multi-use vial, whichallows for repeat administrations (e.g., from 2-6 administrations) ofthe reconstituted formulation. The kit may further comprise a secondcontainer comprising a suitable diluent (e.g., sodium bicarbonatesolution).

Upon mixing of the diluent and the lyophilized formulation, the finalpeptide concentration in the reconstituted formulation is preferably atleast 0.15 mg/mL/peptide (=75 μg) and preferably not more than 3mg/mL/peptide (=1500 μg). The kit may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use.

Kits of the present invention may have a single container that containsthe formulation of the pharmaceutical compositions according to thepresent invention with or without other components (e.g., othercompounds or pharmaceutical compositions of these other compounds) ormay have a distinct container for each component.

Preferably, kits of the invention include a formulation of the inventionpackaged for use in combination with the co-administration of a secondcompound (such as adjuvants (e.g. GM-CSF), a chemotherapeutic agent, anatural product, a hormone or antagonist, an anti-angiogenesis agent orinhibitor, an apoptosis-inducing agent or a chelator) or apharmaceutical composition thereof. The components of the kit may bepre-complexed or each component may be in a separate distinct containerprior to administration to a patient. The components of the kit may beprovided in one or more liquid solutions, preferably, an aqueoussolution, more preferably, a sterile aqueous solution. The components ofthe kit may also be provided as solids, which may be converted intoliquids by addition of suitable solvents, which are preferably providedin another distinct container.

The container of a therapeutic kit may be a vial, test tube, flask,bottle, syringe, or any other means of enclosing a solid or liquid.Usually, when there is more than one component, the kit will contain asecond vial or other container, which allows for separate dosing. Thekit may also contain another container for a pharmaceutically acceptableliquid. Preferably, a therapeutic kit will contain an apparatus (e.g.,one or more needles, syringes, eye droppers, pipette, etc.), whichenables administration of the agents of the invention that arecomponents of the present kit.

The present formulation is one that is suitable for administration ofthe peptides by any acceptable route such as oral (enteral), nasal,ophthal, subcutaneous, intradermal, intramuscular, intravenous ortransdermal. Preferably, the administration is s.c., and most preferablyi.d. administration by infusion pump.

Since the peptides of the invention were isolated from acute myeloidleukemia, breast cancer, cholangiocellular carcinoma, chroniclymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, gastro-esophageal junction cancer,hepatocellular carcinoma, head and neck squamous cell carcinoma,melanoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovariancancer, esophageal cancer, pancreatic cancer, prostate cancer, renalcell carcinoma, small cell lung cancer, urinary bladder carcinoma, anduterine endometrial cancer, the medicament of the invention ispreferably used to treat acute myeloid leukemia, breast cancer,cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectalcancer, gallbladder cancer, glioblastoma, gastric cancer,gastro-esophageal junction cancer, hepatocellular carcinoma, head andneck squamous cell carcinoma, melanoma, non-Hodgkin lymphoma, non-smallcell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, small cell lung cancer, urinarybladder carcinoma, and uterine endometrial cancer.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; and (b)selecting at least one peptide identified de novo in (a) and confirmingits immunogenicity.

Once the peptides for a personalized peptide based vaccine are selected,the vaccine is produced. The vaccine preferably is a liquid formulationconsisting of the individual peptides dissolved in between 20-40% DMSO,preferably about 30-35% DMSO, such as about 33% DMSO.

Each peptide to be included into a product is dissolved in DMSO. Theconcentration of the single peptide solutions has to be chosen dependingon the number of peptides to be included into the product. The singlepeptide-DMSO solutions are mixed in equal parts to achieve a solutioncontaining all peptides to be included in the product with aconcentration of ˜2.5 mg/ml/peptide. The mixed solution is then diluted1:3 with water for injection to achieve a concentration of 0.826mg/ml/peptide in 33% DMSO. The diluted solution is filtered through a0.22 μm sterile filter. The final bulk solution is obtained.

The final bulk solution is filled into vials and stored at −20° C. untiluse. One vial contains 700 μL solution, containing 0.578 mg of eachpeptide. Of this, 500 μL (approx. 400 μg per peptide) will be appliedfor intradermal injection.

In addition to being useful for treating cancer, the peptides of thepresent invention are also useful as diagnostics. Since the peptideswere generated from acute myeloid leukemia, breast cancer,cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectalcancer, gallbladder cancer, glioblastoma, gastric cancer,gastro-esophageal junction cancer, hepatocellular carcinoma, head andneck squamous cell carcinoma, melanoma, non-Hodgkin lymphoma, non-smallcell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, small cell lung cancer, urinarybladder carcinoma, and uterine endometrial cancer cells and since it wasdetermined that these peptides are not or at lower levels present innormal tissues, these peptides can be used to diagnose the presence of acancer.

The presence of claimed peptides on tissue biopsies in blood samples canassist a pathologist in diagnosis of cancer. Detection of certainpeptides by means of antibodies, mass spectrometry or other methodsknown in the art can tell the pathologist that the tissue sample ismalignant or inflamed or generally diseased, or can be used as abiomarker for acute myeloid leukemia, breast cancer, cholangiocellularcarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladdercancer, glioblastoma, gastric cancer, gastro-esophageal junction cancer,hepatocellular carcinoma, head and neck squamous cell carcinoma,melanoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovariancancer, esophageal cancer, pancreatic cancer, prostate cancer, renalcell carcinoma, small cell lung cancer, urinary bladder carcinoma, anduterine endometrial cancer. Presence of groups of peptides can enableclassification or sub-classification of diseased tissues.

The detection of peptides on diseased tissue specimen can enable thedecision about the benefit of therapies involving the immune system,especially if T-lymphocytes are known or expected to be involved in themechanism of action. Loss of MHC expression is a well describedmechanism by which infected or malignant cells escapeimmuno-surveillance. Thus, presence of peptides shows that thismechanism is not exploited by the analyzed cells.

The peptides of the present invention might be used to analyzelymphocyte responses against those peptides such as T cell responses orantibody responses against the peptide or the peptide complexed to MHCmolecules. These lymphocyte responses can be used as prognostic markersfor decision on further therapy steps. These responses can also be usedas surrogate response markers in immunotherapy approaches aiming toinduce lymphocyte responses by different means, e.g. vaccination ofprotein, nucleic acids, autologous materials, adoptive transfer oflymphocytes. In gene therapy settings, lymphocyte responses againstpeptides can be considered in the assessment of side effects. Monitoringof lymphocyte responses might also be a valuable tool for follow-upexaminations of transplantation therapies, e.g. for the detection ofgraft versus host and host versus graft diseases.

The present invention will now be described in the following exampleswhich describe preferred embodiments thereof, and with reference to theaccompanying figures, nevertheless, without being limited thereto. Forthe purposes of the present invention, all references as cited hereinare incorporated by reference in their entireties. Further, note thatexperimental data and figures may only be disclosed herein for aselected set of peptides as claimed. Although for all peptides disclosedand claimed herein, complete data sets have been generated and can bemade available upon request, applicant has decided to not incorporateherein all these complete date sets, because this would go beyond amanageable scope of this application text.

FIGURES

FIG. 1 shows the antigen processing pathway of deamidated peptides andtheir presentation by HLA class I. During translation, proteins with theglycosylation motif N[X{circumflex over ( )}P][ST] become glycosylatedat their N residues in the ER (1). After their export, the cytosolicamidase PNGase removes the N-linked oligosaccharides, resulting in adeamidation to an aspartate (D) (2). After further degradation in theproteasome (3) and re-transported of the peptides into the ER (4), theybind to HLA I complexes (5). The peptide-HLA I complexes are thenfollowing the classical antigen presentation pathway, translocating tothe cell membrane and presenting the deamidated peptides on the cellsurface (6).

FIG. 2 shows the deamidation reactions from asparagine (N) to asparticacid (D) (FIG. 2 ).

FIGS. 3A through 3D show the over-presentation of various peptides indifferent cancer tissues compared to normal tissues. Upper part: MedianMS signal intensities from technical replicate measurements are plottedas dots for single HLA-A*02 positive normal (grey dots, left part offigure) and tumor samples (black dots, right part of figure) on whichthe peptide was detected. Boxes display median, 25th and 75th percentileof normalized signal intensities, while whiskers extend to the lowestdata point still within 1.5 interquartile range (IQR) of the lowerquartile, and the highest data point still within 1.5 IQR of the upperquartile. Lower part: The relative peptide detection frequency in everyorgan is shown as spine plot. Numbers below the panel indicate number ofsamples on which the peptide was detected out of the total number ofsamples analyzed for each organ (N>750 for normal samples, N>675 fortumor samples). If the peptide has been detected on a sample but couldnot be quantified for technical reasons, the sample is included in thisrepresentation of detection frequency, but no dot is shown in the upperpart of the figure. Tissues (from left to right): Normal samples:adipose (adipose tissue); adrenal gl (adrenal gland); bile duct;bladder; bloodcells; bloodvess (blood vessels); bone marrow; brain;breast; esoph (esophagus); eye; gall bl (gallbladder); nead&neck; heart;intest. la (large intestine); intest. sm (small intestine); kidney;liver; lung; lymph nodes; nerve cent (central nerve); nerve periph(peripheral nerve); ovary; pancreas; parathyr (parathyroid gland); perit(peritoneum); pituit (pituitary); placenta; pleura; prostate; skel. mus(skeletal muscle); skin; spinal cord; spleen; stomach; testis; thymus;thyroid; trachea; ureter; uterus. Tumor samples: AML (acute myeloidleukemia); BRCA (breast cancer); CCC (cholangiocellular carcinoma); CLL(chronic lymphocytic leukemia); CRC (colorectal cancer); GBC(gallbladder cancer); GBM (glioblastoma); GC (gastric cancer); GEJC(gastro-esophageal junction cancer); HCC (hepatocellular carcinoma);HNSCC (head and neck squamous cell carcinoma); MEL (melanoma); NHL(non-Hodgkin lymphoma); NSCLCadeno (non-small cell lung canceradenocarcinoma); NSCLCother (NSCLC samples that could not unambiguouslybe assigned to NSCLCadeno or NSCLCsquam); NSCLCsquam (squamous cellnon-small cell lung cancer); OC (ovarian cancer); OSCAR (esophagealcancer); PACA (pancreatic cancer); PRCA (prostate cancer); RCC (renalcell carcinoma); SCLC (small cell lung cancer); UBC (urinary bladdercarcinoma); UEC (uterine endometrial cancer). FIG. 3A) Peptide:ILDSTTIEI (SEQ ID NO: 1), FIG. 3B) Peptide: RLLEGDFSL (SEQ ID NO: 2),FIG. 3C) Peptide: YMDGTMSQV (SEQ ID NO: 3), FIG. 3D) Peptide: YVWDRTELL(SEQ ID NO: 4).

FIGS. 4A through 4D show exemplary expression profile of source genes ofthe present invention that are overexpressed in different cancersamples. Tumor (black dots) and normal (grey dots) samples are groupedaccording to organ of origin. Box-and-whisker plots represent medianFPKM value, 25th and 75th percentile (box) plus whiskers that extend tothe lowest data point still within 1.5 interquartile range (IQR) of thelower quartile and the highest data point still within 1.5 IQR of theupper quartile. FPKM: fragments per kilobase per million mapped reads.Tissues (from left to right): Normal samples: adipose (adipose tissue);adrenal gl (adrenal gland); bile duct; bladder; bloodcells; bloodvess(blood vessels); bone marrow; brain; breast; esoph (esophagus); eye;gall bl (gallbladder); nead&neck; heart; intest. la (large intestine);intest. sm (small intestine); kidney; liver; lung; lymph nodes; nerveperiph (peripheral nerve); ovary; pancreas; parathyr (parathyroidgland); perit (peritoneum); pituit (pituitary); placenta; pleura;prostate; skel. mus (skeletal muscle); skin; spinal cord; spleen;stomach; testis; thymus; thyroid; trachea; ureter; uterus. Tumorsamples: AML (acute myeloid leukemia); BRCA (breast cancer); CCC(cholangiocellular carcinoma); CLL (chronic lymphocytic leukemia); CRC(colorectal cancer); GBC (gallbladder cancer); GBM (glioblastoma); GC(gastric cancer); GEJC (gastro-esophageal junction cancer); HCC(hepatocellular carcinoma); HNSCC (head and neck squamous cellcarcinoma); MEL (melanoma); NHL (non-Hodgkin lymphoma); NSCLCadeno(non-small cell lung cancer adenocarcinoma); NSCLCother (NSCLC samplesthat could not unambiguously be assigned to NSCLCadeno or NSCLCsquam);NSCLCsquam (squamous cell non-small cell lung cancer); OC (ovariancancer); OSCAR (esophageal cancer); PACA (pancreatic cancer); PRCA(prostate cancer); RCC (renal cell carcinoma); SCLC (small cell lungcancer); UBC (urinary bladder carcinoma); UEC (uterine endometrialcancer). FIG. 4A) Ensembl ID: ENST00000263321p369, Peptide: YMNGTMSQV(SEQ ID NO: 105) FIG. 4B) Ensembl ID: ENST00000244763p188, Peptide:SQTNLSPAL (SEQ ID NO: 161), FIG. 4C) Ensembl ID: ENST00000254508p924,Peptide: FISNFTMTI (SEQ ID NO: 163), FIG. 4D) Ensembl ID:ENST00000314191p3305, Peptide: KMLNETVLV (SEQ ID NO: 186).

FIG. 5 shows the results of the IdentControl experiments for oneexemplary peptide AIYHDITGISV (SEQ ID NO: 48). The peptide was confirmedby IdentControl comparing the fragmentations of stable isotope labeled(SIL) standards in data-dependent acquisition (DDA) mode. Identity wasconfirmed using in-house determined spectral correlation threshold.

FIG. 6 shows one exemplary result for a CoElution experiment for thepeptide YMDGTMSQV (SEQ ID NO: 3). The peptide was confirmed by CoElutionusing stable isotope labeled (SIL) internal standard and targeted MS(sPRM or IS-PRM). Non overlapping MS2 isolation windows for theSIL-peptide and the natural peptide are used. Control experiments usingnon-HLA peptidome sample (e.g. tryptic digest or 5% FA) as matrix areperformed to confirm isotopic purity of the SIL internal standard.Peptide identity is confirmed based on objective, predefined criteria inexpert manual review.

FIGS. 7A through 7E show exemplary results of peptide-specific in vitroCD8+ T cell responses of a healthy HLA-A*02+ donor. CD8+ T cells wereprimed using artificial APCs coated with anti-CD28 mAb and HLA-A*02 incomplex with SEQ ID NO 3 (FIG. 7A, left panel), SEQ ID NO 72 (FIG. 7B,left panel), SEQ ID NO 91 (FIG. 7C, left panel), SEQ ID NO 92 (FIG. 7D,left panel) or SEQ ID NO 98 (FIG. 7E, left panel). After three cycles ofstimulation, the detection of peptide-reactive cells was performed by 2Dmultimer staining with A*02/SEQ ID NO 3 (FIG. 7A), A*02/SEQ ID NO 6(FIG. 7B), A*02/SEQ ID NO 7 (FIG. 7C), A*02/SEQ ID NO 47 (FIG. 7D) orA*02/SEQ ID NO 96 (FIG. 7E). Right panels (FIGS. 7A, 7B, 7C, 7D and 7E)show control staining of cells stimulated with irrelevant A*02/peptidecomplexes. Viable single cells were gated for CD8+ lymphocytes. Booleangates helped excluding false-positive events detected with multimersspecific for different peptides. Frequencies of specific multimer+ cellsamong CD8+ lymphocytes are indicated.

EXAMPLES Example 1

Identification and Quantitation of Tumor Associated Peptides Presentedon the Cell Surface

Tissue Samples

Patients' tissues were obtained from:

BioIVT (Detroit, MI, USA & Royston, Herts, UK); Bio-Options Inc. (Brea,CA, USA); BioServe (Beltsville, MD, USA); Capital BioScience Inc.(Rockville, MD, USA); Conversant Bio (Huntsville, AL, USA); CurelineInc. (Brisbane, CA, USA); DxBiosamples (San Diego, CA, USA); GeneticistInc. (Glendale, CA, USA); Indivumed GmbH (Hamburg, Germany); KyotoPrefectural University of Medicine (KPUM) (Kyoto, Japan); Osaka CityUniversity (OCU) (Osaka, Japan); ProteoGenex Inc. (Culver City, CA,USA); Tissue Solutions Ltd (Glasgow, UK); Universitst Bonn (Bonn,Germany); Asklepios Clinic St. Georg (Hamburg, Germany); Val d'HebronUniversity Hospital (Barcelona, Spain); Center for cancer immune therapy(CCIT), Herlev Hospital (Herlev, Denmark); Leiden University MedicalCenter (LUMC) (Leiden, Netherlands); Istituto Nazionale Tumori“Pascale”, Molecular Biology and Viral Oncology Unit (Naples, Italy);Stanford Cancer Center (Palo Alto, CA, USA); University Hospital Geneva(Geneva, Switzerland); University Hospital Heidelberg (Heidelberg,Germany); University Hospital Munich (Munich, Germany); UniversityHospital Tuebingen (Tuebingen, Germany).

Written informed consents of all patients had been given before surgeryor autopsy. Tissues were shock-frozen immediately after excision andstored until isolation of TUMAPs at −70° C. or below.

Isolation of HLA Peptides from Tissue Samples

HLA peptide pools from shock-frozen tissue samples were obtained byimmune precipitation from solid tissues according to a slightly modifiedprotocol (Falk et al., 1991; Seeger et al., 1999) using the HLA-A*02specific antibody BB7.2, the HLA-A, -B, -C specific antibody W6/32, theHLA-DR specific antibody L243 and the HLA-DP specific antibody B7/21,CNBr-activated sepharose, acid treatment, and ultrafiltration.

All peptides according to the present invention bind to HLA-A*02.However due to similarity in binding patterns, e.g. in the anchoringpositions, the peptides may also bind to other HLA alleles whichinclude, but are not limited to, HLA-B*08, HLA-B*13 and HLA-B*15.

Furthermore, the term HLA-A*02 refers to all subtypes of HLA-A*02, whichinclude but are not limited to HLA-A*02:01, HLA-A*02:02, HLA-A*02:03,HLA-A*02:04, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, HLA-A*02:08,HLA-A*02:09, HLA-A*02:10, HLA-A*02:11.

Mass Spectrometry Analyses

The HLA peptide pools as obtained were separated according to theirhydrophobicity by reversed-phase chromatography (nanoAcquity UPLCsystem, Waters) and the eluting peptides were analyzed in LTQ velos andfusion hybrid mass spectrometers (ThermoElectron) equipped with an ESIsource. Peptide pools were loaded directly onto the analyticalfused-silica micro-capillary column (75 μm i.d.×250 mm) packed with 1.7μm C18 reversed-phase material (Waters) applying a flow rate of 400 nLper minute. Subsequently, the peptides were separated using a two-step180 minute-binary gradient from 10% to 33% B at a flow rate of 300 nLper minute. The gradient was composed of Solvent A (0.1% formic acid inwater) and solvent B (0.1% formic acid in acetonitrile). A gold coatedglass capillary (PicoTip, New Objective) was used for introduction intothe nanoESI source. The LTQ-Orbitrap mass spectrometers were operated inthe data-dependent mode using a TOP5 strategy. In brief, a scan cyclewas initiated with a full scan of high mass accuracy in the orbitrap(R=30000), which was followed by MS/MS scans also in the orbitrap(R=7500) on the 5 most abundant precursor ions with dynamic exclusion ofpreviously selected ions. Tandem mass spectra were interpreted bySEQUEST at a fixed false discovery rate (qs0.05) and additional manualcontrol. In cases where the identified peptide sequence was uncertain itwas additionally validated by comparison of the generated naturalpeptide fragmentation pattern with the fragmentation pattern of asynthetic sequence-identical reference peptide.

Label-free relative LC-MS quantitation was performed by ion countingi.e. by extraction and analysis of LC-MS features (Mueller et al.,2007). The method assumes that the peptide's LC-MS signal areacorrelates with its abundance in the sample. Extracted features werefurther processed by charge state deconvolution and retention timealignment (Mueller et al., 2008; Sturm et al., 2008). Finally, all LC-MSfeatures were cross-referenced with the sequence identification resultsto combine quantitative data of different samples and tissues to peptidepresentation profiles. The quantitative data were normalized in atwo-tier fashion according to central tendency to account for variationwithin technical and biological replicates. Thus, each identifiedpeptide can be associated with quantitative data allowing relativequantification between samples and tissues. In addition, allquantitative data acquired for peptide candidates was inspected manuallyto assure data consistency and to verify the accuracy of the automatedanalysis. For each peptide a presentation profile was calculated showingthe mean sample presentation as well as replicate variations. Theprofiles juxtapose AML (acute myeloid leukemia); BRCA (breast cancer);CCC (cholangiocellular carcinoma); CLL (chronic lymphocytic leukemia);CRC (colorectal cancer); GBC (gallbladder cancer); GBM (glioblastoma);GC (gastric cancer); GEJC (gastro-esophageal junction cancer); HCC(hepatocellular carcinoma); HNSCC (head and neck squamous cellcarcinoma); MEL (melanoma); NHL (non-Hodgkin lymphoma); NSCLCadeno(non-small cell lung cancer adenocarcinoma); NSCLCother (NSCLC samplesthat could not unambiguously be assigned to NSCLCadeno or NSCLCsquam);NSCLCsquam (squamous cell non-small cell lung cancer); OC (ovariancancer); OSCAR (esophageal cancer); PACA (pancreatic cancer); PRCA(prostate cancer); RCC (renal cell carcinoma); SCLC (small cell lungcancer); UBC (urinary bladder carcinoma); UEC (uterine endometrialcancer) samples to a baseline of normal tissue samples. Presentationprofiles of exemplary over-presented peptides are shown in FIGS. 3A-3D.The plots show only those identifications of peptides as dots which weremade on HLA-A*02 positive tissue samples which were processed usingHLA-A*02 specific antibodies.

Peptide presentation on the various indications for all peptides (SEQ IDNO: 1 to SEQ ID NO: 102) are shown in table 8. This table lists allindication on which the respective peptide was identified at least once,independent of the HLA typing of the sample or the antibody used toprocess said sample.

TABLE 8 Presentation on various cancer entities for peptidesaccording to the invention, and thus the particular relevanceof the peptides as mentioned for  the diagnosis and/ortreatment of the cancers as indicated. SEQ ID NO SEQUENCEPEPTIDE PRESENTATION ON CANCER TYPES 1 ILDSTTIEINHL, OC, AML, UBC, OSCAR, NSCLCsquam,NSCLCother, NSCLCadeno, MEL, HNSCC, SCLC,HCC, GC, GBC, CRC, BRCA, UEC, RCC, PACA, GEJC, CLL 2 RLLEGDFSLUEC, OSCAR, OC, NSCLCsquam, NSCLCother,HNSCC, HCC, GBC, CRC, NSCLCadeno, BRCA 3 YMDGTMSQV MEL, NHL 4 YVWDRTELLUEC 5 ALPQFFNDV GC, RCC, GBC, HCC, PACA, UBC, PRCA,OSCAR, NSCLCadeno, BRCA, OC, NSCLCother, MEL, GEJC, AML 6 NLYNDLTNVHNSCC, CRC, NSCLCsquam, NSCLCadeno,BRCA, SCLC, PACA, OSCAR, OC, NHL, HCC, CCC, UEC, MEL, GEJC, GC, GBC 7ALADLTGTV HCC, MEL, AML, UEC, SCLC, RCC, UBC,OSCAR, OC, NSCLCsquam, NSCLCother,NSCLCadeno, GBM, GBC, CRC, CLL, CCC, BRCA 8 KLAPDLTELNSCLCadeno, GBM, PRCA, RCC, PACA, CRC,BRCA, UEC, SCLC, OSCAR, NSCLCsquam, NSCLCother, HCC, CCC 9 SLDDSLNELPACA, MEL, GBC, OSCAR, OC, CRC, BRCA,UEC, UBC, PRCA, NSCLCadeno, NHL, HCC, AML 10 FLGEDISNFLUBC, UEC, RCC, NSCLCadeno, GC, SCLC,PACA, OC, NSCLCsquam, NSCLCother, MEL, HCC, CCC 11 GLVDGSSVTVRCC, NHL, OC, NSCLCsquam, HCC, NSCLCother, CRC, CCC, BRCA 12 FVDEHTIDIGBC, PACA, OC, MEL, UBC, RCC, PRCA, NHL, BRCA 13 VLMDGTLKQVHCC, CCC, SCLC 14 YIYDGKDMSSL OSCAR, HCC, NHL, MEL, CRC, UBC, PRCA, OC,NSCLCsquam, GEJC, GBM 15 VLDDTLVIFGBC, GC, UBC, RCC, PACA, OC, NHL, MEL, HCC, BRCA 16 KLISDMGKDVSAHNSCC, OSCAR, NSCLCsquam, NSCLCother 17 NVDSTILNLPACA, GBC, UBC, OSCAR, OC, NSCLCadeno, GC, BRCA 18 ILDEAKDISFGBC, PACA, UBC, PRCA, OC, MEL 19 RLLDTTDVYLLOC, SCLC, NSCLCsquam, NSCLCadeno, UEC, OSCAR 20 SLFAYPLPDVSCLC, NSCLCsquam, NHL, MEL, OC, NSCLCother, GBM, CRC, AML 21 FLNLFDHTLMEL, NSCLCsquam, CRC, CLL, BRCA, NSCLCadeno, HNSCC 22 HMIDLTIHLUBC, NSCLCadeno, NSCLCother, MEL, BRCA 23 FLGPIVDLNHL, CLL, AML, OC, NSCLCadeno 24 QLLGRDISLCLL, BRCA, OC, NSCLCother, NHL, MEL, GC, GBM, AML 25 FMNDRSFILSCLC, CRC, BRCA, PACA, NSCLCsquam, GC, GBC 26 ILYDGSNIQLRCC, PRCA, OSCAR, HNSCC 27 YLFEDISQL HNSCC, OC, NSCLCadeno 28FLLDGTDMFHM HNSCC, UBC, OC 29 KLLDLTVRI CRC, PACA, NSCLCsquam, GC, GBC30 QLMEKVQDV GBM, UEC, HNSCC, GBC 31 TLIDATWLVNSCLCadeno, HNSCC, NSCLCother 32 ALADLTGTVVNLUEC, RCC, PRCA, OC, NSCLCadeno, HCC, CRC 33 ILFDLTHRVRCC, NSCLCadeno, OSCAR, HNSCC, GC 34 SLLDGSESAKL OC, MEL, HNSCC, BRCA 35SLRDGTEVV HNSCC, OSCAR, NSCLCsquam, NSCLCother, NHL 36 SLYDFTGEQMAAOC, NSCLCother, NSCLCadeno, NHL, CCC 37 AIDDTAARLPACA, OC, HCC, GC, GBC, AML 38 AVDGTDARL RCC, GC, OC 39 AVFDQTVTVIPRCA, OSCAR, MEL, HCC, GBM 40 FLDQTDETL BRCA, UEC 41 FLDTSTADVNHL, AML, CLL 42 IQDTSHLAV RCC, OC, NSCLCsquam, NHL, GC, CRC 43KLLNDLTSI MEL, HCC, NSCLCsquam, NHL 44 LLDATHQIHCC, RCC, NSCLCsquam, MEL, CRC 45 SLFDDSFSLUBC, NSCLCsquam, HNSCC, CRC, CCC, BRCA 46 SLSDVSQAVAML, NSCLCadeno, NHL, MEL, BRCA 47 VIDSTLVKV NSCLCsquam, MEL, GBM 48AIYHDITGISV NSCLCadeno, OC, NSCLCsquam 49 ALDDYTITF GC, UBC, HCC, AML 50ALYDSTRELL NSCLCadeno, BRCA, NSCLCsquam 51 HLCNHDVSLBRCA, NSCLCadeno, CRC 52 ILFDDSVFTL NHL, HNSCC, HCC, CLL, AML 53ILTDITGHDV OC, NSCLCother, NSCLCadeno, NHL, GBM 54 KLEERVYDVUBC, NSCLCsquam, MEL, BRCA 55 KTWDQSIAL HCC, OSCAR, CCC 56 LLDSTAHLLPACA, OC, MEL, GC, GBM 57 SLDISLPNL UEC, PRCA, CLL, BRCA 58 SQDASLLKVRCC, OSCAR, NHL, GC, CLL 59 SQTDLSPAL HCC, NSCLCsquam, CCC 60 TLDITIVNLBRCA, RCC, OC, GBC 61 FISDFTMTI MEL, SCLC 62 FLKDVTAQINHL, NSCLCsquam, MEL 63 GLDRTQLVNV RCC, UBC, NSCLCadeno 64 ILFDPDNSSALRCC, HCC, GC, CCC 65 ILLDKSTVL UBC, HNSCC, CRC 66 LLDDQTVTF PACA, OC, GC67 LLDTTDVYL UEC, SCLC, OC, MEL 68 RLQDTTIGL CRC 69 SLLDENDVSSYLNHL, CLL 70 SMASFLKDV HNSCC, NSCLCsquam 71 YLGAVFDLUBC, SCLC, OC, NHL, CRC 72 ALDSTNSEL CLL, NHL 73 ALMDVSQNVNSCLCsquam, HNSCC 74 KILDFTGPLFL CLL, NSCLCsquam 75 KLHDTSFCL CRC, HCC76 SLLRDFTLV RCC, NHL, GC 77 YLHGFDLSL HNSCC 78 AIDQTITEA UEC, MEL 79ALAGLVYDA UEC, NHL 80 ALDSSLQLL NHL, AML 81 AVDKTQTSV SCLC, CRC 82FLNPDGSDCTL NHL, OC, AML, UBC, OSCAR, NSCLCsquam,NSCLCother, NSCLCadeno, MEL, HNSCC, SCLC,HCC, GC, GBC, CRC, BRCA, UEC, RCC, PACA, GEJC, CLL 83 FLNVDVSEVNSCLCadeno, MEL 84 KMLDETVLV MEL 85 LLDITNPVL UEC, CLL 86 NLADNTILVUBC, HCC 87 NMYDLTFHV NHL 88 RLLDETVDVTI NSCLCsquam, NSCLCadeno 89SLFIDVTRV HCC, GC 90 VLDGTEVNL MEL 91 VLKDGTLVI MEL, GEJC 92 YLADFSHATNHL, AML 93 YLDGTFRLL OC, BRCA 94 YLVVWVNDQSL CRC 95 KLAPEDLADLTALAML, BC, CLL, HCC, HNSCC, MEL, NHL,NSCLCsquam, NSCLCadeno, NSCLCother, SCLC 96 KLDASLPALNSCLCadeno, CRC, OC, NSCLCsquam, NSCLCother, HNSCC, HCC, NHL, MEL, BRCA,OSCAR, AML, UEC, UBC, RCC, GBC, PACA, GEJC, GBM, SCLC, PRCA, GC, CCC 97KLLDGSQRV HNSCC, PACA, OC, NSCLCsquam, HCC, BRCA,UEC, SCLC, OSCAR, NSCLCother, MEL 98 NLLADVSTV OC, NSCLCadeno 99NLLDHSSMFL OC, NSCLCsquam, HCC, BRCA, RCC,NSCLCadeno, CRC, CLL, AML, OSCAR, UEC,PACA, NSCLCother, NHL, MEL, GC, CCC 100 SLLFQDITL AML, OC 101 TLDATVVELUBC, HNSCC, BRCA, OC, OSCAR, UEC, PACA, GC, GBC 102 WLIDKSMELNSCLCadeno, OC, NSCLCsquam, NSCLCother,CRC, MEL, UEC, SCLC, OSCAR, NHL, HNSCC, HCC, GC, BRCA, RCC, CCC, GBCCancer type: AML (acute myeloid leukemia); BRCA (breast cancer); CCC(cholangiocellular carcinoma); CLL (chronic lymphocytic leukemia); CRC(colorectal cancer); GBC (gallbladder cancer); GBM (glioblastoma); GC(gastric cancer); GEJC (gastro-esophageal junction cancer); HCC(hepatocellular carcinoma); HNSCC (head and neck squamous cellcarcinoma); MEL (melanoma); NHL (non-Hodgkin lymphoma); NSCLCadeno(non-small cell lung cancer adenocarcinoma); NSCLCother (NSCLC samplesthat could not unambiguously be assigned to NSCLCadeno or NSCLCsquam);NSCLCsquam (squamous cell non-small cell lung cancer); OC (ovariancancer); OSCAR (esophageal cancer); PACA (pancreatic cancer); PRCA(prostate cancer); RCC (renal cell carcinoma); SCLC (small cell lungcancer); UBC (urinary bladder carcinoma); UEC (uterine endometrialcancer).

Example 2

Expression Profiling of Genes Encoding the Peptides of the Invention

Over-presentation or specific presentation of a peptide on tumor cellscompared to normal cells is sufficient for its usefulness inimmunotherapy, and some peptides are tumor-specific despite their sourceprotein occurring also in normal tissues. Still, mRNA expressionprofiling adds an additional level of safety in selection of peptidetargets for immunotherapies. Especially for therapeutic options withhigh safety risks, such as affinity-matured TCRs, the ideal targetpeptide will be derived from a protein that is unique to the tumor andnot found on normal tissues.

RNA Sources and Preparation

Surgically removed tissue specimens were provided as indicated above(see Example 1) after written informed consent had been obtained fromeach patient. Tumor tissue specimens were snap-frozen immediately aftersurgery and later homogenized with mortar and pestle under liquidnitrogen. Total RNA was prepared from these samples using TRI Reagent(Ambion, Darmstadt, Germany) followed by a cleanup with RNeasy (QIAGEN,Hilden, Germany); both methods were performed according to themanufacturer's protocol.

Total RNA from healthy human tissues for RNASeq experiments was obtainedfrom: Asterand (Detroit, MI, USA & Royston, Herts, UK); Bio-Options Inc.(Brea, CA, USA); Geneticist Inc. (Glendale, CA, USA); ProteoGenex Inc.(Culver City, CA, USA); Tissue Solutions Ltd (Glasgow, UK).

Total RNA from tumor tissues for RNASeq experiments was obtained from:Asterand (Detroit, MI, USA & Royston, Herts, UK); BioCat GmbH(Heidelberg, Germany); BioServe (Beltsville, MD, USA); Geneticist Inc.(Glendale, CA, USA); Istituto Nazionale Tumori “Pascale” (Naples,Italy); ProteoGenex Inc. (Culver City, CA, USA); University HospitalHeidelberg (Heidelberg, Germany).

Quality and quantity of all RNA samples were assessed on an Agilent 2100Bioanalyzer (Agilent, Waldbronn, Germany) using the RNA 6000 PicoLabChip Kit (Agilent).

RNAseq Experiments

Gene expression analysis of tumor and normal tissue RNA samples wasperformed by next generation sequencing (RNAseq) by CeGaT (Tübingen,Germany). Briefly, sequencing libraries are prepared using the IlluminaHiSeq v4 reagent kit according to the provider's protocol (IlluminaInc., San Diego, CA, USA), which includes RNA fragmentation, cDNAconversion and addition of sequencing adaptors. Libraries derived frommultiple samples are mixed equimolar and sequenced on the Illumina HiSeq2500 sequencer according to the manufacturer's instructions, generating50 bp single end reads. Processed reads are mapped to the human genome(GRCh38) using the STAR software. Expression data are provided ontranscript level as RPKM (Reads Per Kilobase per Million mapped reads,generated by the software Cufflinks) and on exon level (total reads,generated by the software Bedtools), based on annotations of the ensemblsequence database (Ensembl77). Exon reads are normalized for exon lengthand alignment size to obtain RPKM values.

Exemplary expression profiles of source genes of the present inventionthat are highly overexpressed or exclusively expressed in AML (acutemyeloid leukemia); BRCA (breast cancer); CCC (cholangiocellularcarcinoma); CLL (chronic lymphocytic leukemia); CRC (colorectal cancer);GBC (gallbladder cancer); GBM (glioblastoma); GC (gastric cancer); GEJC(gastro-esophageal junction cancer); HCC (hepatocellular carcinoma);HNSCC (head and neck squamous cell carcinoma); MEL (melanoma); NHL(non-Hodgkin lymphoma); NSCLCadeno (non-small cell lung canceradenocarcinoma); NSCLCother (NSCLC samples that could not unambiguouslybe assigned to NSCLCadeno or NSCLCsquam); NSCLCsquam (squamous cellnon-small cell lung cancer); OC (ovarian cancer); OSCAR (esophagealcancer); PACA (pancreatic cancer); PRCA (prostate cancer); RCC (renalcell carcinoma); SCLC (small cell lung cancer); UBC (urinary bladdercarcinoma); UEC (uterine endometrial cancer) are shown in FIGS. 4A-4D.Expression scores for further exemplary genes are shown in table 9.

TABLE 9 Expression scores.The table lists peptides from genes that are highlyoverexpressed in tumors compared to a panel of normal tissues (++) oroverexpressed in tumors compared to a panel of normal tissues (+).The baseline for this score was calculated from measurements of thefollowing relevant normal tissues: adipose tissue; adrenal gland;bile duct; bladder; bloodcells; blood vessels; bone marrow; brain;breast; esophagus; eye; gallbladder; nead&neck; heart; large intestine;small intestine; kidney; liver; lung; lymph nodes; peripheral nerve;ovary; pancreas; parathyroid gland; peritoneum; pituitary; placenta;pleura; prostate; skeletal muscle; skin; spinal cord; spleen; stomach;testis; thymus; thyroid; trachea; ureter; uterus. In case expressiondata for several samples of the same tissue type were available, thearithmetic mean of all respective samples was used for the calculation.GENE EXPRESSION IN TUMORS HIGHLY SEQ ID NO SEQUENCE OVEREPRESSED (+)OVEREXPRESSED (++) 104 RLLEGNFSL HNSCC, OSCAR, SCLC UEC 105 YMNGTMSQVMEL 106 YVWNRTELL CRC, GBC, GC, NSCLCadeno, NSCLCother, OC, OSCAR,PACA, UEC 126 QLLGRNISL AML 129 YLFENISQL HNSCC 139 AINDTAARLHNSCC, OSCAR 150 AIYHNITGISV NSCLCadeno NSCLCother 161 SQTNLSPAL HCC 163FISNFTMTI MEL 167 ILLNKSTVL MEL 186 KMLNETVLV MEL 189 NMYNLTFHV NHL 190RLLNETVDVTI UBC 191 SLFINVTRV CCC 193 VLKNGTLVI PACA 196 YLWWVNNQSL CRC203 TLNATVVEL UBC

Example 3

Validation of Peptides by IdentControl and CoElution

In order to validate the peptides according to the invention, allpeptides were synthesized using standard and well-established solidphase peptide synthesis using the Fmoc-strategy. If necessary, stableisotope labeled (SIL-) amino acids were used to introduce adiscriminating mass shift and allow for the use of these labeledpeptides as internal standards (e.g. if a peptide was selected foridentity confirmation in CoElution experiments). Identity and purity ofeach individual peptide were determined by mass spectrometry andanalytical RP-HPLC. The peptides were obtained as white to off-whitelyophilizes (trifluoro acetate salt) in purities of >50%. All TUMAPs arepreferably administered as trifluoro-acetate salts or acetate salts,other salt-forms are also possible.

The initial validation of peptides was achieved by IdentControl viaspectral comparison. For this, synthetic peptides were used forvalidation of peptide identifications by acquisition of high-resolutionreference MS2 spectra using matched fragmentation modes and collisionenergies as for acquisition of the natural spectra. Automated spectralcomparison was performed using the sensitive metric of spectralcorrelation with a cutoff score determined to result in 90% sensitivityat <1% FDR based on a benchmark dataset comprising >10,000 manuallyvalidated spectra. Ambiguous identifications were further subjected tovalidation in CoElution experiments.

TABLE 10 IdentControl Results.The spectral correlation indicates the similarityof the MS/MS spectra from the endogenous detectedpeptide compared to the synthetic peptide, thehigher the value the more alike the spectra are.The peptide is validated when a threshold of0.75 is met, or spectra are considered identical by manual review.SEQ ID NO SEQUENCE SPECTRAL CORRELATION 1 ILDSTTIEI 0.925 2 RLLEGDFSL0.989 3 YMDGTMSQV 0.991 4 YVWDRTELL 0.941 5 ALPQFFNDV 0.984 6 NLYNDLTNV0.913 7 ALADLTGTV 0.924 8 KLAPDLTEL 0.851 9 SLDDSLNEL 0.933 10FLGEDISNFL 0.980 11 GLVDGSSVTV 0.885 12 FVDEHTIDI 0.951 13 VLMDGTLKQV0.774 14 YIYDGKDMSSL 0.932 15 VLDDTLVIF 0.972 16 KLISDMGKDVSA 0.839 17NVDSTILNL 0.950 18 ILDEAKDISF 0.936 19 RLLDTTDVYLL 0.802 20 SLFAYPLPDV0.951 21 FLNLFDHTL 0.951 22 HMIDLTIHL 0.959 23 FLGPIVDL 0.827 24QLLGRDISL 0.920 25 FMNDRSFIL 0.945 26 ILYDGSNIQL 0.839 27 YLFEDISQL0.965 28 FLLDGTDMFHM 0.877 29 KLLDLTVRI 0.988 30 QLMEKVQDV 0.900 31TLIDATWLV 0.785 32 ALADLTGTVVNL 0.843 33 ILFDLTHRV 0.943 34 SLLDGSESAKL0.879 35 SLRDGTEVV 0.961 36 SLYDFTGEQMAA 0.969 37 AIDDTAARL 0.947 38AVDGTDARL 0.952 39 AVFDQTVTVI 0.956 40 FLDQTDETL 0.900 41 FLDTSTADV0.951 42 IQDTSHLAV 0.833 43 KLLNDLTSI 0.773 44 LLDATHQI 0.811 45SLFDDSFSL 0.804 46 SLSDVSQAV 0.792 47 VIDSTLVKV 0.935 48 AIYHDITGISV0.992 49 ALDDYTITF 0.879 50 ALYDSTRELL 0.973 51 HLCNHDVSL 0.964 52ILFDDSVFTL 0.909 53 ILTDITGHDV 0.952 54 KLEERVYDV 0.847 55 KTWDQSIAL0.848 56 LLDSTAHLL 0.968 57 SLDISLPNL 0.989 58 SQDASLLKV 0.931 59SQTDLSPAL 0.906 60 TLDITIVNL 0.860 61 FISDFTMTI 0.985 62 FLKDVTAQI 0.94463 GLDRTQLVNV 0.977 64 ILFDPDNSSAL 0.856 65 ILLDKSTVL 0.964 66 LLDDQTVTF0.929 67 LLDTTDVYL 0.953 68 RLQDTTIGL 0.916 69 SLLDENDVSSYL 0.866 70SMASFLKDV 0.897 71 YLGAVFDL 0.987 72 ALDSTNSEL 0.820 73 ALMDVSQNV 0.91274 KILDFTGPLFL 0.963 75 KLHDTSFCL 0.838 76 SLLRDFTLV 0.974 77 YLHGFDLSL0.847 78 AIDQTITEA 0.976 79 ALAGLVYDA 0.984 80 ALDSSLQLL 0.889 81AVDKTQTSV 0.954 82 FLNPDGSDCTL 0.866 83 FLNVDVSEV 0.961 84 KMLDETVLV0.964 85 LLDITNPVL 0.925 86 NLADNTILV 0.972 87 NMYDLTFHV 0.916 88RLLDETVDVTI 0.876 89 SLFIDVTRV 0.823 90 VLDGTEVNL 0.805 91 VLKDGTLVI0.866 92 YLADFSHAT 0.776 93 YLDGTFRLL 0.954 94 YLWWVNDQSL 0.800 95KLAPEDLADLTAL 0.971 96 KLDASLPAL 0.739 97 KLLDGSQRV 0.871 98 NLLADVSTV0.565 99 NLLDHSSMFL 0.948 100 SLLFQDITL 0.625 101 TLDATVVEL 0.913 102WLIDKSMEL 0.915

For further validation peptides were subjected to CoElution experimentsusing SIL internal standard peptides. To this end, SIL peptides werespiked into HLA peptidome extracts from samples and subjected to liquidchromatography—targeted mass spectrometry (LC-MS) to confirm peptideidentity based on spectral similarity as well as CoElution in theretention time dimension. Spiked SIL-peptide amounts were adjusted tothe peptide specific ionization factors (determined in calibrationcurves), if necessary. LC-MS was performed using pre-defined targetedMS2 scan events with non-overlapping isolation windows for SIL-peptideand natural peptide species to avoid co-fragmentation. To confirmisotopic purity and absence of co-fragmentation of SIL- and naturalpeptide, control experiments were performed in a non-HLA peptidecontaining tryptic matrix, which had to confirm absence of any unlabeledsignal. Peptide detection and validation by CoElution was determined bymanual expert review based on multiple pre-defined objective criteria,including dot product (dotP) of SIL peptide compared to unlabeledpeptide MS2 traces, the presence of the most intense transitions inmultiple consecutive scans and aligned peak apexes. A list whichpeptides were validated by CoElution can be found in table 11.

TABLE 11 Peptides with positive CoElution experiment SEQ ID NO SEQUENCE1 ILDSTTIEI 3 YMDGTMSQV 5 ALPQFFNDV 6 NLYNDLTNV 7 ALADLTGTV 9 SLDDSLNEL10 FLGEDISNFL 11 GLVDGSSVTV 12 FVDEHTIDI 13 VLMDGTLKQV 19 RLLDTTDVYLL 20SLFAYPLPDV 21 FLNLFDHTL 23 FLGPIVDL 24 QLLGRDISL 25 FMNDRSFIL 27YLFEDISQL 29 KLLDLTVRI 32 ALADLTGTVVNL 33 ILFDLTHRV 34 SLLDGSESAKL 37AIDDTAARL 41 FLDTSTADV 43 KLLNDLTSI 46 SLSDVSQAV 47 VIDSTLVKV 48AIYHDITGISV 49 ALDDYTITF 50 ALYDSTRELL 52 ILFDDSVFTL 53 ILTDITGHDV 57SLDISLPNL 59 SQTDLSPAL 63 GLDRTQLVNV 67 LLDTTDVYL 71 YLGAVFDL 72ALDSTNSEL 73 ALMDVSQNV 74 KILDFTGPLFL 77 YLHGFDLSL 78 AIDQTITEA 79ALAGLVYDA 80 ALDSSLQLL 81 AVDKTQTSV 82 FLNPDGSDCTL 83 FLNVDVSEV 84KMLDETVLV 85 LLDITNPVL 86 NLADNTILV 87 NMYDLTFHV 89 SLFIDVTRV 90VLDGTEVNL 92 YLADFSHAT 93 YLDGTFRLL

Example 4

Absolute Quantitation of Tumor Associated Peptides Presented on the CellSurface

The generation of binders, such as antibodies and/or TCRs, is alaborious process, which may be conducted only for a number of selectedtargets. In the case of tumor associated and specific peptides,selection criteria include, but are not restricted to, exclusiveness ofpresentation and the density of peptide presented on the cell surface.In addition to the isolation and relative quantitation of peptides asdescribed in EXAMPLE 1, the inventors did analyze absolute peptidecopies per cell as described in WO 2016/107740. The quantitation ofTUMAP copies per cell in solid tumor samples requires the absolutequantitation of the isolated TUMAP, the efficiency of the TUMAPisolation process, and the cell count of the tissue sample analyzed.

Peptide Quantitation by Nano LC-MS/MS

For an accurate quantitation of peptides by mass spectrometry, acalibration curve was generated for each individual peptide using twodifferent isotope labeled peptide variants (one or two isotope-labeledamino acids are included during TUMAP synthesis). These isotopes labeledvariants differ from the tumor-associated peptide only in their mass butshow no difference in other physicochemical properties (Anderson et al.,2012). For the peptide calibration curve, a series of nano LC-MS/MSmeasurements was performed to determine the ration of MS/MS signals oftitrated (singly isotope-labeled peptide) to constant (doubly isotopelabeled peptide) isotope labeled peptides.

The doubly isotope labeled peptide, also called internal standard, wasfurther spiked to each MS sample and all MS signals were normalized tothe MS signal of the internal standard to level out potential technicalvariances between MS experiments.

The calibration curves were prepared in at least three differentmatrices, i.e. HLA peptide eluates from natural samples similar to theroutine MS samples, and each preparation was measured in duplicate MSruns. For evaluation, MS signals were normalized to the signal of theinternal standard and a calibration curve was calculated by logisticregression.

For the quantitation of tumor-associated peptides from tissue samples,the respective samples were also spiked with the internal standard; theMS signals were normalized to the internal standard and quantified usingthe peptide calibration curve.

Efficiency of Peptide-MHC Isolation

As for any protein purification process, the isolation of proteins fromtissue samples is associated with a certain loss of the protein ofinterest. To determine the efficiency of TUMAP isolation, peptide-MHCcomplexes were generated for all TUMAPs selected for absolutequantitation. To be able to discriminate the spiked from the naturalpeptide-MHC complexes, single-isotope-labelled versions of the TUMAPswere used, i.e. one isotope-labelled amino acid was included in TUMAPsynthesis. These complexes were spiked into the freshly prepared tissuelysates, i.e. at the earliest possible point of the TUMAP isolationprocedure, and then captured like the natural peptide-MHC complexes inthe following affinity purification. Measuring the recovery of thesingle-labelled TUMAPs therefore allows conclusions regarding theefficiency of isolation of individual natural TUMAPs.

The efficiency of isolation was analyzed in a small set of samples andwas comparable among these tissue samples. In contrast, the isolationefficiency differs between individual peptides. This suggests that theisolation efficiency, although determined in only a limited number oftissue samples, may be extrapolated to any other tissue preparation.However, it is necessary to analyze each TUMAP individually as theisolation efficiency may not be extrapolated from one peptide to others.

Determination of the Cell Count in Solid, Frozen Tissue

In order to determine the cell count of the tissue samples subjected toabsolute peptide quantitation, the inventors applied DNA contentanalysis. This method is applicable to a wide range of samples ofdifferent origin and, most importantly, frozen samples (Alcoser et al.,2011; Forsey and Chaudhuri, 2009; Silva et al., 2013). During thepeptide isolation protocol, a tissue sample is processed to a homogenouslysate, from which a small lysate aliquot is taken. The aliquot isdivided in three parts, from which DNA is isolated (QiaAmp DNA Mini Kit,Qiagen, Hilden, Germany). The total DNA content from each DNA isolationis quantified using a fluorescence-based DNA quantitation assay (QubitdsDNA HS Assay Kit, Life Technologies, Darmstadt, Germany) in at leasttwo replicates.

In order to calculate the cell number, a DNA standard curve fromaliquots of isolated healthy blood cells from several donors, with arange of defined cell numbers, has been generated. The standard curve isused to calculate the total cell content from the total DNA content fromeach DNA isolation. The mean total cell count of the tissue sample usedfor peptide isolation is then extrapolated considering the known volumeof the lysate aliquots and the total lysate volume.

Peptide Copies Per Cell

With data of the aforementioned experiments, the inventors calculatedthe number of TUMAP copies per cell by dividing the total peptide amountby the total cell count of the sample, followed by division throughisolation efficiency. Copy cell number for selected peptides are shownin table 12.

A more elaborate disclosure of the method to absolutely quantify thepeptides is disclosed in international patent publication WO2016107740A1and U.S. patent application Ser. No. 14/969,423, the contents of both ofwhich is incorporated herein by reference.

TABLE 12 Absolute copy numbers of peptides according to the invention.The table lists the results of absolute peptidequantitation in tumor samples. The median numberof copies per cell are indicated for each peptide:≥25 = +; ≥50 = ++; ≥75 = +++, ≥100 = ++++The number of samples, in which evaluablehigh-quality MS data are available, is indicated. COPIES PER CELLNUMBER OF SEQ ID NO SEQUENCE (MEDIAN) SAMPLES 2 RLLEGDFSL + 2 61FISDFTMTI ++++ 2

Example 5

In Vitro Immunogenicity for MHC Class I Presented Peptides

In order to obtain information regarding the immunogenicity of theTUMAPs of the present invention, the inventors performed investigationsusing an in vitro T cell priming assay based on repeated stimulations ofCD8+ T cells with artificial antigen presenting cells (aAPCs) loadedwith peptide/MHC complexes and anti-CD28 antibody. This way theinventors could show immunogenicity for HLA-A*02 restricted TUMAPs ofthe invention, demonstrating that these peptides are T cell epitopesagainst which CD8+ precursor T cells exist in humans (Table 13).

In Vitro Priming of CD8+ T Cells

In order to perform in vitro stimulations by artificial antigenpresenting cells loaded with peptide-MHC complex (pMHC) and anti-CD28antibody, the inventors first isolated CD8+ T cells from fresh HLA-A*02leukapheresis products via positive selection using CD8 microbeads(Miltenyi Biotec, Bergisch-Gladbach, Germany) of healthy donors obtainedfrom the University clinics Mannheim, Germany, after informed consent.

PBMCs and isolated CD8+ lymphocytes were incubated in T cell medium(TCM) until use consisting of RPMI-Glutamax (Invitrogen, Karlsruhe,Germany) supplemented with 10% heat inactivated human AB serum(PAN-Biotech, Aidenbach, Germany), 100 U/ml Penicillin/100 μg/mlStreptomycin (Cambrex, Cologne, Germany), 1 mM sodium pyruvate (CC Pro,Oberdorla, Germany), 20 μg/ml Gentamycin (Cambrex). 2.5 ng/ml IL-7(PromoCell, Heidelberg, Germany) and 10 U/ml IL-2 (Novartis Pharma,Nürnberg, Germany) were also added to the TCM at this step.

Generation of pMHC/anti-CD28 coated beads, T cell stimulations andreadout was performed in a highly defined in vitro system using fourdifferent pMHC molecules per stimulation condition and 8 different pMHCmolecules per readout condition.

The purified co-stimulatory mouse IgG2a anti human CD28 Ab 9.3 (Jung etal., 1987) was chemically biotinylated usingSulfo-N-hydroxysuccinimidobiotin as recommended by the manufacturer(Perbio, Bonn, Germany). Beads used were 5.6 μm diameter streptavidincoated polystyrene particles (Bangs Laboratories, Illinois, USA).

pMHC used for positive and negative control stimulations wereA*02:01/MLA-001 (peptide ELAGIGILTV (SEQ ID NO: 205) from modifiedMelan-A/MART-1) and A*02:01/DDX5-001 (YLLPAIVHI from DDX5, SEQ ID NO:206), respectively.

800.000 beads/200 μl were coated in 96-well plates in the presence of4×12.5 ng different biotin-pMHC, washed and 600 ng biotin anti-CD28 wereadded subsequently in a volume of 200 μl. Stimulations were initiated in96-well plates by co-incubating 1×10⁶ CD8+ T cells with 2×10⁵ washedcoated beads in 200 μl TCM supplemented with 5 ng/ml IL-12 (PromoCell)for 3 days at 37° C. Half of the medium was then exchanged by fresh TCMsupplemented with 80 U/ml IL-2 and incubating was continued for 4 daysat 37° C. This stimulation cycle was performed for a total of threetimes. For the pMHC multimer readout using 8 different pMHC moleculesper condition, a two-dimensional combinatorial coding approach was usedas previously described (Andersen et al., 2012) with minor modificationsencompassing coupling to 5 different fluorochromes. Finally, multimericanalyses were performed by staining the cells with Live/dead near IR dye(Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1 (BD,Heidelberg, Germany) and fluorescent pMHC multimers. For analysis, a BDLSRII SORP cytometer equipped with appropriate lasers and filters wasused. Peptide specific cells were calculated as percentage of total CD8+cells. Evaluation of multimeric analysis was done using the FlowJosoftware (Tree Star, Oregon, USA). In vitro priming of specificmultimer+ CD8+ lymphocytes was detected by comparing to negative controlstimulations. Immunogenicity for a given antigen was detected if atleast one evaluable in vitro stimulated well of one healthy donor wasfound to contain a specific CD8+ T cell line after in vitro stimulation(i.e. this well contained at least 1% of specific multimer+ among CD8+ Tcells and the percentage of specific multimer+ cells was at least 10×the Median of the Negative Control stimulations).

In Vitro Immunogenicity for Cancer Peptides

For tested HLA class I peptides, in vitro immunogenicity could bedemonstrated by generation of peptide specific T cell lines. Exemplaryflow cytometry results after TUMAP-specific multimer staining for 5peptides of the invention are shown in FIGS. 7A-7E together withcorresponding negative controls. Results for 12 peptides from theinvention are summarized in Table 13.

TABLE 13 in vitro immunogenicity ofHLA class I peptides of the inventionExemplary results of in vitro immunogenicityexperiments conducted by the applicant forHLA-A*02 restricted peptides of the invention.Results of in vitro immunogenicity experiments areindicated. Percentage of positive wells and donors(among evaluable) are summarized as indicated<20% = +; 20%-49% = ++; 50%-69% = +++; >=70% = ++++ SEQ ID NO SequenceWells positive [%] 3 YMDGTMSQV +++ 6 NLYNDLTNV + 7 ALADLTGTV + 8KLAPDLTEL + 47 VIDSTLVKV ++ 65 ILLDKSTVL + 76 SLLRDFTLV + 96 KLDASLPAL +97 KLLDGSQRV + 98 NLLADVSTV + 100 SLLFQDITL + 101 TLDATVVEL ++

Example 6

MHC Binding Assays

Candidate peptides for T cell based therapies according to the presentinvention were further tested for their MHC binding capacity (affinity).The individual peptide-MHC complexes were produced by UV-ligandexchange, where a UV-sensitive peptide is cleaved upon UV-irradiationand exchanged with the peptide of interest as analyzed. Only peptidecandidates that can effectively bind and stabilize the peptide-receptiveMHC molecules prevent dissociation of the MHC complexes. To determinethe yield of the exchange reaction, an ELISA was performed based on thedetection of the light chain (β2m) of stabilized MHC complexes. Theassay was performed as generally described in Rodenko et al. (Rodenko etal., 2006).

96 well MAXISorp plates (NUNC) were coated over night with 2 μg/mlstreptavidin in PBS at room temperature, washed 4× and blocked for 1 hat 37° C. in 2% BSA containing blocking buffer. RefoldedHLA-A*02:01/MLA-001 monomers served as standards, covering the range of15-500 ng/ml. Peptide-MHC monomers of the UV-exchange reaction werediluted 100-fold in blocking buffer. Samples were incubated for 1 h at37° C., washed four times, incubated with 2 μg/ml HRP conjugatedanti-β2m for 1h at 37° C., washed again and detected with TMB solutionthat is stopped with NH₂SO₄. Absorption was measured at 450 nm.Candidate peptides that show a high exchange yield (preferably higherthan 50%, most preferred higher than 75%) are generally preferred for ageneration and production of antibodies or fragments thereof, and/or Tcell receptors or fragments thereof, as they show sufficient avidity tothe MHC molecules and prevent dissociation of the MHC complexes.

MHC-peptide binding results for 102 peptides from the invention aresummarized in Table 14.

TABLE 14 MHC class I binding scores.Binding of HLA-class I restricted peptides toHLA-A*02:01 was ranged by peptide exchange yield:≥10% = +; ≥20% = ++; ≥50% = +++; ≥75% = ++++ SEQ ID NO SequencePeptide exchange 1 ILDSTTIEI ++++ 2 RLLEGDFSL ++++ 3 YMDGTMSQV +++ 4YVWDRTELL ++++ 5 ALPQFFNDV ++++ 6 NLYNDLTNV +++ 7 ALADLTGTV ++++ 8KLAPDLTEL +++ 9 SLDDSLNEL +++ 10 FLGEDISNFL ++++ 11 GLVDGSSVTV ++++ 12FVDEHTIDI +++ 13 VLMDGTLKQV ++++ 14 YIYDGKDMSSL +++ 15 VLDDTLVIF +++ 16KLISDMGKDVSA +++ 17 NVDSTILNL ++++ 18 ILDEAKDISF +++ 19 RLLDTTDVYLL ++++20 SLFAYPLPDV ++++ 21 FLNLFDHTL ++++ 22 HMIDLTIHL +++ 23 FLGPIVDL ++++24 QLLGRDISL ++++ 25 FMNDRSFIL ++++ 26 ILYDGSNIQL ++++ 27 YLFEDISQL ++++28 FLLDGTDMFHM +++ 29 KLLDLTVRI ++++ 30 QLMEKVQDV +++ 31 TLIDATWLV ++++32 ALADLTGTVVNL ++++ 33 ILFDLTHRV ++++ 34 SLLDGSESAKL ++++ 35 SLRDGTEVV+++ 36 SLYDFTGEQMAA +++ 37 AIDDTAARL +++ 38 AVDGTDARL +++ 39 AVFDQTVTVI+++ 40 FLDQTDETL +++ 41 FLDTSTADV ++++ 42 IQDTSHLAV +++ 43 KLLNDLTSI++++ 44 LLDATHQI ++++ 45 SLFDDSFSL ++++ 46 SLSDVSQAV +++ 47 VIDSTLVKV+++ 48 AIYHDITGISV +++ 49 ALDDYTITF ++++ 50 ALYDSTRELL ++++ 51 HLCNHDVSL++++ 52 ILFDDSVFTL ++++ 53 ILTDITGHDV +++ 54 KLEERVYDV ++++ 55 KTWDQSIAL++++ 56 LLDSTAHLL ++++ 57 SLDISLPNL ++++ 58 SQDASLLKV +++ 59 SQTDLSPAL++++ 60 TLDITIVNL ++++ 61 FISDFTMTI ++++ 62 FLKDVTAQI ++++ 63 GLDRTQLVNV++++ 64 ILFDPDNSSAL ++++ 65 ILLDKSTVL ++++ 66 LLDDQTVTF +++ 67 LLDTTDVYL+++ 68 RLQDTTIGL ++++ 69 SLLDENDVSSYL ++++ 70 SMASFLKDV ++++ 71 YLGAVFDL++++ 72 ALDSTNSEL ++++ 73 ALMDVSQNV ++++ 74 KILDFTGPLFL ++++ 75KLHDTSFCL ++++ 76 SLLRDFTLV ++++ 77 YLHGFDLSL ++++ 78 AIDQTITEA +++ 79ALAGLVYDA ++++ 80 ALDSSLQLL ++++ 81 AVDKTQTSV +++ 82 FLNPDGSDCTL ++++ 83FLNVDVSEV ++++ 84 KMLDETVLV +++ 85 LLDITNPVL ++++ 86 NLADNTILV ++++ 87NMYDLTFHV ++++ 88 RLLDETVDVTI ++++ 89 SLFIDVTRV ++++ 90 VLDGTEVNL ++++91 VLKDGTLVI ++ 92 YLADFSHAT ++++ 93 YLDGTFRLL +++ 94 YLWWVNDQSL ++++ 95KLAPEDLADLTAL ++++ 96 KLDASLPAL ++++ 97 KLLDGSQRV +++ 98 NLLADVSTV +++99 NLLDHSSMFL ++++ 100 SLLFQDITL ++++ 101 TLDATVVEL ++++ 102 WLIDKSMEL++++

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What is claimed is:
 1. A peptide consisting of the amino acid sequenceof ILDSTTIEI (SEQ ID NO: 1) in the form of a pharmaceutically acceptablesalt.
 2. The peptide according to claim 1, wherein said peptide has theability to bind to an MHC class 1 molecule, and/or wherein said peptide,when bound to said MHC, is capable of being recognized by CD4 and/or CD8T cells.
 3. The peptide of claim 1, wherein the pharmaceuticallyacceptable salt is chloride salt.
 4. The peptide of claim 1, wherein thepharmaceutically acceptable salt is acetate salt.
 5. The peptide ofclaim 1, wherein the pharmaceutically acceptable salt istrifluoro-acetate salt.
 6. The peptide of claim 1, wherein said peptideis produced by solid phase peptide synthesis or produced by a yeast cellor bacterial cell expression system.
 7. A pharmaceutical compositioncomprising the peptide according to claim 1 and a pharmaceuticallyacceptable carrier.
 8. The pharmaceutical composition of claim 7,further comprises an adjuvant.
 9. The pharmaceutical composition ofclaim 8, wherein the adjuvant is selected from the group consisting ofanti-CD40 antibody, imiquimod, resiquimod, GM-CSF, cyclophosphamide,sunitinib, bevacizumab, interferon-alpha, interferon-beta, CpGoligonucleotides and derivatives, poly-(I:C) and derivatives, RNA,sildenafil, particulate formulations with poly(lactide co-glycolide)(PLG), virosomes, interleukin (IL)-1, IL-2, IL-4, IL-7, IL-12, IL-13,IL-15, IL-21, and IL-23.
 10. The pharmaceutical composition of claim 9,wherein the adjuvant is IL-1.
 11. The pharmaceutical composition ofclaim 9, wherein the adjuvant is IL-2.
 12. The pharmaceuticalcomposition of claim 9, wherein the adjuvant is IL-4.
 13. Thepharmaceutical composition of claim 9, wherein the adjuvant is IL-7. 14.The pharmaceutical composition of claim 9, wherein the adjuvant isIL-12.
 15. The pharmaceutical composition of claim 9, wherein theadjuvant is IL-13.
 16. The pharmaceutical composition of claim 9,wherein the adjuvant is IL-15.
 17. The pharmaceutical composition ofclaim 9, wherein the adjuvant is IL-21.
 18. The pharmaceuticalcomposition of claim 9, wherein the adjuvant is IL-23.
 19. Thepharmaceutical composition of claim 7, wherein the pharmaceuticallyacceptable carrier is selected from saline, Ringer's solution, dextrosesolution, and sustained release preparation.
 20. The pharmaceuticalcomposition of claim 7, further comprising water and a buffer.